Analysis of Ubiquitinated Polypeptides

ABSTRACT

The invention relates to antibody reagents that specifically bind to peptides carrying a ubiquitin remnant from a digested or chemically treated biological sample. The reagents allow the technician to identify ubiquitinated polypeptides as well as the sites of ubiquitination on them. The reagents are preferably employed in proteomic analysis 
     using mass spectrometry. The antibody reagents specifically bind to the remnant of ubiquitin (i.e., a diglycine modified epsilon amine of lysine) left on a peptide which as been generated by digesting or chemically treating ubiquitinated proteins. The inventive antibody reagents&#39; affinity to the ubiquitin remnant does not depend on the remaining amino acid sequences flanking the modified (i.e., ubiquitinated) lysine, i.e., they are context independent.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/967,824 filed Dec.14, 2010, which claims benefit from U.S. provisional patent applicationSer. No. 61/286,486 filed Dec. 15, 2009, pending, the entire disclosuresof which are hereby incorporated by reference. U.S. Ser. No. 12/967,824,filed Dec. 14, 2010 is also a continuation-in-part of U.S. Ser. No.11/823,775 filed Jun. 28, 2007, pending, which itself is a divisionalapplication of U.S. Ser. No. 10/777,893, filed Feb. 12, 2004, now U.S.Pat. No. 7,300,753, which itself is a continuation-in-part of U.S. Ser.No. 10/175,486, filed Jun. 19, 2002, now U.S. Pat. No. 7,198,896, whichitself claims priority to U.S. Ser. No. 60/299,893, filed Jun. 21, 2001,and U.S. Ser. No. 60/337,012, filed Nov. 8, 2001, both expired, theentire disclosures of each of which are hereby incorporated byreference.

FIELD OF THE INVENTION

This invention provides methods, reagents and kits for analyzingpolypeptides and their modifications from biological samples. Inparticular, the invention provides compositions, kits and methods fordetecting ubiquitinated polypeptides and ubiquitination sites inproteins.

BACKGROUND OF THE INVENTION

Personalized medicine is the application of genomic and molecular datato better target the delivery of health care to specific patients,facilitate the discovery and clinical testing of new products, and helpdetermine a person's predisposition to a particular disease orcondition.

On a technical level, personalized medicine depends on theidentification and detection of proteins, genes and genetic variation(“biomarkers”) that play a role in a given disease. Rodland, ClinBiochem. 2004 July; 37(7):579-83. The presence or absence of certainbiomarkers is then correlated with the incidence of a particular diseaseor disease predisposition. However, currently available methods forbiomarker analysis are associated with long waiting periods, high costand numerous technical hurdles.

The current standard for protein detection and/or quantification isbased on immunoreactive detection (Western analysis). However, thistechnique requires the availability of an appropriately specificantibody. In addition, many antibodies only recognize proteins in anunfolded (denatured) form, cross-reactivity can be severely limiting,and quantification is generally relative.

The development of methods and instrumentation for automated,data-dependent electrospray ionization (ESI) tandem mass spectrometry(MS/MS) in conjunction with microcapillary liquid chromatography (LC)and database searching has significantly increased the sensitivity andspeed of the identification of gel-separated proteins. MicrocapillaryLC-MS/MS has been used successfully for the large-scale identificationof individual proteins directly from mixtures without gelelectrophoretic separation (Link et al., 1999; Opitek et al., 1997).However, while these approaches accelerate protein identification,quantities of the analyzed proteins cannot be easily determined, andthese methods have not been shown to substantially alleviate the dynamicrange problem also encountered by the 2DE/MS/NIS approach. Therefore,low abundance proteins in complex samples are also difficult to analyzeby the microcapillary LC/MS/MS method without their prior enrichment.

Protein ubiquitination is the one of the most common of allpost-translational modifications. Ubiquitin is a highly conserved 76amino acid protein which is linked to a protein target after a cascadeof transfer reactions. Ubiquitin is activated through the formation of athioester bond between its C-terminal glycine and the active sitecysteine of the ubiquitin activating protein, E1 (Hershko, 1991, TrendsBiochem. Sci. 16(7): 265-8). In subsequent trans-thiolation reactions,Ubiquitin is transferred to a cysteine residue on a ubiquitinconjugating enzyme, E2 (Hershko, et al., 1983, J. Biol. Chem. 267:8807-8812). In conjunction with E3, a ubiquitin polypeptide ligase, E2then transfers ubiquitin to a specific polypeptide target (see, e.g.,Scheffner, et al., 1995, Nature 373(6509): 81-3), forming an isopeptidebond between the C-terminal glycine of ubiquitin and the 8-amino groupof a lysine present in the target (See FIG. 1).

The covalent attachment of ubiquitin to cellular polypeptides, in mostcases, marks them for degradation by a multi-polypeptide complex calleda proteosome. The ubiquitinproteosome system is the principal mechanismfor the turnover of short-lived polypeptides, including regulatorypolypeptides (Weissman, 2001, Nat. Rev. Mol. Cell. Biol. 2: 169-78).Some known targets of ubiquitination include: cyclins, cyclin-dependentkinases (CDK's), NFKB, cystic fibrosis transduction receptor, p53,ornithine decarboxylase (ODC), 7-membrane spanning receptors, Cdc25(phosphotyrosme phosphatase), Rb, Ga, c-Jun and c-Fos. Polypeptidessharing consensus sequences such as PEST sequences, destruction boxes,and F-boxes generally are also targets for ubiquitin-mediateddegradation pathways (see, e.g., Rogers, et al., 1986, Science 234:364-368; Yamano, et al., 1998, The EMBO Journal 17: 5670-5678; Bai, etal., 1996, Cell 86: 263-274).

Ubiquitin has been implicated in a number of cellular processesincluding: signal transduction, cell-cycle progression,receptor-mediated endocytosis, transcription, organelle biogenesis,spermatogenesis, response to cell stress, DNA repair, differentiation,programmed cell death, and immune responses (e.g., inflammation).Ubiquitin also has been implicated in the biogenesis of ribosomes,nucleosomes, peroxisomes and myofibrils. Thus, ubiquitin can functionboth as signal for polypeptide degradation and as a chaperone forpromoting the formation of organelles (see, e.g., Fujimuro, et al.,1997, Eur. J. Biochem. 249: 427-433).

Deregulation of ubiquitination has been implicated in the pathogenesisof many different diseases. For example, abnormal accumulations ofubiquitinated species are found in patients with neurodegenerativediseases such as Alzheimer's as well as in patients with cellproliferative diseases, such as cancer (see, e.g., Hershko andCiechanover, 1998, Annu Rev. Biochem. 67: 425-79; Layfield, et al.,2001, Neuropathol. Appl. Neurobiol. 27:171-9; Weissman, 1997, ImmunologyToday 18(4): 189).

While the importance of its biological role is well appreciated, theubiquitin pathway is inherently difficult to study. Generally, studiesof ubiquitination have focused on particular polypeptides. For example,site-directed mutagenesis has been used to evaluate critical amino acidswhich form the “destruction boxes”, or “D-boxes”, of cyclin, sites whichare rapidly poly-ubiquitinated when cyclin is triggered for destruction.See, e.g., Yamano, et al., 1998, The EMBO Journal 17: 5670-5678; Amon etal., 1994, Cell 77: 1037-1050; Glotzer, et al., 1991, Nature 349:132-138; King, et al., 1996, Mol. Biol. Cell 7:1343. Corsi, et al.,1997, J. Biol. Chem. 272(5): 2977-2883, which describe a Westernblotting approach to identify ubiquitination sites. In this technique,crude radiolabeled a-spectrin fractions were ubiquitinated in vitro,digested with proteases, and electrophoresed on gels. Ubiquitinatedpeptides were identified by their differences in mass from peptidesgenerated by digestion of non-ubiquitinated α-spectrin.

Although mass spectrometry offers a powerful tool for identifyingubiquitin substrates, a number of unresolved issues remain. Despite manyadvances, MS data is inherently biased toward more abundant substrates.The effects of ubiquitin epitope tags used to enriched ubiqunatedproteins remain incompletely understood, including whether purificationbiases exist and whether ubiquitin pathway enzymes utilize tagged andwild-type ubiquitin with equal efficiency. It is also not clear ifubiquitin-binding proteins or ubiquitin antibodies may work efficientlyas affinity reagents in order to lessen the need for epitope.Kirkpatrick et al., Nat Cell Biol. 2005 August; 7(8): 750-757.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a method for determining thepresence of at least one ubiquitinated polypeptide in a biologicalsample comprising: Contacting the sample with at least one hydrolyzingagent, wherein the hydrolyzing agent is capable of cleaving aubiquitinated polypeptide to produce at least one ubiquitin remnantpeptide, to obtain a hydrolyzed sample; Contacting the hydrolyzed samplewith a substrate comprising an at least one immobilized binding partner;wherein the at least one immobilized binding partner preferentiallybinds a ubiquitin remnant peptide over a peptide having the same aminoacid sequence as the ubiquitin remnant peptide but lacking a ubiquitinremnant; Removing the hydrolyzed sample from the substrate in a mannersuch that the at least one ubiquitin remnant peptide would remain boundto the immobilized binding partner; Contacting the substrate with anelution solution, wherein the least one ubiquitin remnant peptide woulddissociate from the immobilized binding partner into the elutionsolution; and Determining the presence of a least one ubiquitinatedpolypeptide in the biological sample when the elution solution containsthe at least one least ubiquitin remnant peptide.

In one embodiment of this aspect of the invention the determining isperformed by LC, MS and preferably LC-MS/MS. In a further embodiment,the amino acid sequence of at least one ubiquitin remnant peptidepresent in the elution solution, is determined. In yet anotherembodiment, the sequence is compared to the sequence of theubiquitinated polypeptide and the site of ubiquitination in theubiquitinated polypeptide is thereby determined. In still a furtherembodiment, the elution solution further comprises at least one standardpeptide, wherein the at least one standard peptide has the substantiallythe same amino acid sequence as the at least one distinct peptide but adifferent measured accurate mass.

Another aspect of the invention relates to an isolated antibody thatpreferentially binds a ubiquitin remnant peptide over a peptide havingthe same amino acid sequence as the ubiquitin remnant peptide butlacking a ubiquitin remnant. In one embodiment, the antibody is amonoclonal antibody. In another embodiment, the antibody is a polyclonalantibody. In still yet another embodiment, the antibody is selected fromthe group consisting of single chain Fvs (scFvs), Fab fragments, Fab′fragments, F(ab′) 2, disulfide linked Fvs (sdFvs), Fvs, and fragmentsthereof. In yet another embodiment, the antibody comprises a polypeptideof SEQ ID NO: 1. In a further embodiment, the antibody comprises apolypeptide of SEQ ID NO: 2. In yet another embodiment, the antibodycomprises a light chain polypeptide of SEQ ID NO: 2 and a heavy chainpolypeptide of SEQ ID NO: 1. In still another embodiment, the antibodycomprises an antigen binding site comprising the variable region of theheavy chain set forth in SEQ ID NO: 1. In still a further embodiment,the antibody comprises an antigen binding site comprising the variableregion of the light chain set forth in SEQ ID NO: 2.

Another aspect of the invention relates to an isolated nucleic acidencoding an antibody that preferentially binds a ubiquitin remnantpeptide over a peptide having the same amino acid sequence as theubiquitin remnant peptide but lacking a ubiquitin remnant.

A further aspect of the invention relates to a cell comprising a nucleicacid, preferably in the form of a vector, that encodes an antibody thatpreferentially binds a ubiquitin remnant peptide over a peptide havingthe same amino acid sequence as the ubiquitin remnant peptide butlacking a ubiquitin remnant.

Another aspect of the invention relates to the isolated ubiquitinremnant peptides listed in Table 4 and fragments and variants thereof.

Another aspect of the invention relates to nucleic acids encoding theubiquitin remnant peptides listed in Table 4 and fragments and variantsthereof.

Yet a further aspect of the invention relates to a method fordetermining whether a patient is has or is likely to have or develop adisease associated with a least one ubiquitinated polypeptidecomprising: obtaining a biological sample from the patient; Contactingthe sample with at least one hydrolyzing agent, wherein the hydrolyzingagent is capable of cleaving a ubiquitinated polypeptide to produce atleast one ubiquitin remnant peptide, to obtain a hydrolyzed sample;Contacting the hydrolyzed sample with a substrate comprising an at leastone immobilized binding partner; wherein the at least one immobilizedbinding partner preferentially binds a ubiquitin remnant peptide over apeptide having the same amino acid sequence as the ubiquitin remnantpeptide but lacking a ubiquitin remnant; Removing the hydrolyzed samplefrom the substrate in a manner such that the at least one ubiquitinremnant peptide would remain bound to the immobilized binding partner;Contacting the substrate with an elution solution, where in the leastone ubiquitin remnant peptide would dissociate from the immobilizedbinding partner into the elution solution; and Determining the presenceof a least one ubiquitinated polypeptide in the biological sample whenthe elution solution contains the at least one least ubiquitin remnantpeptide; Determining that the patient is has or is likely to have ordevelop the disease associated with a least one ubiquitinatedpolypeptide if the least one ubiquitinated polypeptide is present in thebiological sample.

Another aspect of the invention relates to a method for determiningwhether a disease is associated with at least one ubiquitinatedpolypeptide comprising Obtaining a biological sample from a patienthaving the disease; Contacting the sample with at least one hydrolyzingagent, wherein the hydrolyzing agent is capable of cleaving aubiquitinated polypeptide to produce at least one ubiquitin remnantpeptide, to obtain a hydrolyzed sample; Contacting the hydrolyzed samplewith a substrate comprising an at least one immobilized binding partner;wherein the at least one immobilized binding partner preferentiallybinds a ubiquitin remnant peptide over a peptide having the same aminoacid sequence as the ubiquitin remnant peptide but lacking a ubiquitinremnant; Removing the hydrolyzed sample from the substrate in a mannersuch that the at least one ubiquitin remnant peptide would remain boundto the immobilized binding partner; Contacting the substrate with anelution solution, where in the least one ubiquitin remnant peptide woulddissociate from the immobilized binding partner into the elutionsolution; Determining the presence of a least one ubiquitinatedpolypeptide in the biological sample when the elution solution containsthe at least one least ubiquitin remnant peptide; and Determining thatthe disease is associated with the presence of the at least oneubiquitinated polypeptide if the least one ubiquitinated polypeptide isabsent in the biological sample of a healthy individual.

Still another aspect of the invention relates to a method fordetermining whether a disease is associated with at least one ubiquitinremnant peptide Obtaining a biological sample from a patient having thedisease to obtain a disease biological sample; Obtaining a biologicalsample from a healthy patient to obtains a healthy biological sample;Contacting the disease biological sample with at least one hydrolyzingagent, wherein the hydrolyzing agent is capable of cleaving aubiquitinated polypeptide to produce the least one ubiquitin remnantpeptide, to obtain a disease hydrolyzed sample; Contacting the healthybiological sample with at least one hydrolyzing agent, wherein thehydrolyzing agent is capable of cleaving a ubiquitinated polypeptide toproduce the least one ubiquitin remnant peptide, to obtain a healthyhydrolyzed sample; Contacting the disease hydrolyzed sample with asubstrate comprising an at least one immobilized binding partner;wherein the at least one immobilized binding partner preferentiallybinds a ubiquitin remnant peptide over a peptide having the same aminoacid sequence as the ubiquitin remnant peptide but lacking a ubiquitinremnant; Removing the disease hydrolyzed sample from the substrate in amanner such that the at least one ubiquitin remnant peptide would remainbound to the immobilized binding partner; Contacting the substrate withan elution solution, where in the least one ubiquitin remnant peptidewould dissociate from the immobilized binding partner into the elutionsolution; and Determining the presence of the a least one ubiquitinremnant peptide in the elution solution; Determining that the disease isassociated with the presence of the at least one ubiquitin remnantpeptide if the least one ubiquitin remnant peptide is absent in thehealthy biological sample.

BRIEF DESCRIPTION OF THE FIGURES

A more complete understanding of the present invention may be obtainedby reference to the accompanying drawings, when considered inconjunction with the subsequent detailed description. The embodimentsillustrated in the drawings are intended only to exemplify the inventionand should not be construed as limiting the invention to the illustratedembodiments, in which:

FIG. 1 depicts a cartoon of the formation of a ubiquitin remnant

FIG. 2 shows a heat map illustrating the frequency of amino acids foundwith the BL4936 polyclonal antibody in a study of four mouse tissues.Altogether 1458 non-redundant peptides were included in this frequencymap. The map clearly shows there are no strongly preferred amino acidsat least seven residues to the amino-terminal side of K(GG) modificationsites (−7 to −1 in the figure) or at least seven residues to thecarboxyl-terminal side of K(GG) modification sites.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered antibody reagents that specifically bindpeptides carrying a ubiquitin remnant from a digested or chemicallytreated biological sample. See also U.S. application Ser No. 12/455,496(which is incorporated by reference in its entirety for all purposes andwithout limitation).

These reagents allow the technician to identify ubiquitinatedpolypeptides as well as the sites of ubiquitination on them. Thereagents are preferably employed in proteomic analysis using massspectrometry. The antibody reagents (in both polyclonal and monoclonalform) specifically bind the remnant of ubiquitination, i.e., a diglycinemodified epsilon amine of lysine left on a peptide which as beengenerated by digesting or chemically treating ubiquitinated proteins.The inventive antibody reagents' affinity to the ubiquitin remnant doesnot depend on the remaining amino acid sequences flanking the modifiedlysine, i.e., they are “context independent”. In addition, theantibodies of the invention do not cross react with peptides lacking theubiquitin remnant. See for example, U.S. Pat. Nos. 6,441,140; 6,982,318;7,198,896; 7,259,022; 7,300,753; 7,344,714; U.S. Ser. No. 11,484,485,all herein incorporated by reference in their entirety.

Notwithstanding the low abundance ofubiquitinated polypeptides inbiological samples, the invention allows for high-throughput MSidentification of ubiquitination sites. Immunoaffinity purification(IAP) with the inventive antibodies enrich those ubiquitinated peptidesderived from the ubiquitinated portion ofpolypeptides relative topeptides lacking ubiquitination sites, as well as peptides from proteinswhich strongly interact with ubiquitin or ubiquitinated proteins,thereby significantly reducing the complexity of the peptide mixture.The purified digest sample can be directly applied to tandem MS forefficient peptide sequence analysis and protein identification to revealubiquitinated polypeptides and their sites of ubiquitination.

Prior to describing various embodiments of the current invention, thefollowing definitions are provided:

As used herein the term “peptide” or “polypeptide” refers to a polymerformed from the linking, in a defined order, of preferably, α-aminoacids, D-, L-amino acids, and combinations thereof. The link between oneamino acid residue and the next is referred to as an amide bond or apeptide bond. Proteins are polypeptide molecules (or having multiplepolypeptide subunits). The distinction is that peptides are preferablyshort and polypeptides/proteins are preferably longer amino acid chains.The term “protein” is intended to also encompass derivatized moleculessuch as glycoproteins and lipoproteins as well as lower molecular weightpolypeptides.

As used herein, the term “ubiquitinated polypeptide” refers to apolypeptide bound to ubiquitin, a ubiquitin-like protein (e.g., NEDD8 orISG15) or a portion thereof. Preferably, ubiquitination is the formationan isopeptide bond between the C-terminal glycine of ubiquitin (orubiquitin-like protein see e.g., J Proteome Res. 2008 March;7(3):1274-87) and the 8-amino group of a lysine present in the target.(See e.g., FIG. 1).

As used herein, a “ubiquitin remnant” or a “ubiquitin tag” is thatportion of a ubiquitinated polypeptide which remains attached to thedigestion pmduct of the ubiquitinated polypeptide which has been exposedto a hydrolyzing agent such as trypsin. Preferably, the ubiquitinremnant is a diglycine modified epsilon amine of lysine, which addsabout 114 daltons to the mass of the lysine residue (see FIG. 1). It isalso referred to herein as “K(GG).” Trypsin digestion of neddylatedproteins leaves the same K(GG) remnant as trypsin digestion of proteinthat is attached to ubiquitin.

A “ubiquitin remnant peptide” is the product that results from thedigestion of a ubiquitinated polypeptide with a hydrolyzing agent suchas trypsin, i.e., a peptide containing at least one ubiquitin remnant.In the preferred embodiment of the invention, a binding partner is usedthat specifically recognizes and binds to a ubiquitin remnant peptidebut does not cross react with other peptides having the same amino acidsequence but which lack the ubiquitin remnant. The preferred bindingpartner is an antiubiquitin remnant peptide antibody or fragmentthereof.

The invention also encompasses the novel ubiquitin remnant peptidesdisclosed herein in Table 4 as well as fragments and variants thereof.

The term “variant” as used herein relative to ubiquitin remnantpeptides, refers to a peptide having a ubiquitin remnant that possessesa similar or identical amino acid sequence as a ubiquitin remnantpeptide (e.g., one disclosed in Table 4). A variant having a similaramino acid sequence refers to a peptide comprising, or alternativelyconsisting of, an amino acid sequence that is at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identical to the predicate ubiquitin remnant peptide. Peptidevariants also include those having a deletion, substitution and/oraddition of about 1 to about 2; about 1 to about 3; or about 1 to about4 amino acids relative to the predicate ubiquitin remnant peptide.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide at the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity numberof identical overlapping positions/total number ofpositions.times.100%). In one embodiment, the two sequences are the samelength.

The term “fragment” as used herein refers to a peptide comprising aubiquitin remnant and an amino acid sequence of at least 3 amino acidresidues, at least 5 amino acid residues, at least 7 amino acidresidues, at least 10 amino acid residues, at least 15 amino acidresidues, at least 20 amino acid residues, at least 25 amino acidresidues, at least 30 amino acid residues of a ubiquitin remnantpeptide.

The invention also includes nucleic acids that encode for the ubiquitinremnant peptides disclosed herein in Table 4 as well as fragments andvariants thereof.

As used herein, the term “biological sample” refers to a readilyobtainable mixture of a plurality ofpolypeptides present in varyingconcentrations. Preferred biological samples have about 5,000 to about20,000 different polypeptides. More preferably, biological samples haveabout 7,500 to about 15,000 different polypeptides. Most preferably,biological samples have about 10,000 different polypeptides. Generally,such samples are environmental, industrial, veterinary or medical inorigin and from an animal, plant, a bacterium, a fungus, a protist or avirus. The preferred biological samples include but are not limited tosaliva, mucous, tears, blood, serum, lymph/interstitial fluids, buccalcells, mucosal cells, cerebrospinal fluid, semen, feces, plasma, urine,a suspension of cells, or a suspension of cells and viruses. The mostpreferred biological samples are mammalian, more preferably human, serumand urine.

Where the biological sample is blood, serum or lymph/interstitial fluid,the invention envisages an optional step of depleting the biologicalsample of common and disproportionally over-represented backgroundproteins not suspected of being associated with ubiquitinatedpolypeptides. Such proteins include but are not limited to albumin, IgG,IgA, transferrin, haptoglobin, and anti-trypsin; or combinationsthereof. The skilled artisan will recognized that such a step is carriedout by basic affinity chromatography techniques. As used here in theterm “depleted” or “depleting” means markedly lessening theconcentration of a particular species in a solution, e.g., by more thanor about 50%; more than or about 60%; more than or about 65%; more thanor about 70%; more than or about 75%; more than or about 80%; more thanor about 85%; more than or about 90%; more than or about 92%; more thanor about 95%; more than or about 97%; more than or about 98%; more thanor about 99%. Alternatively the biological sample may be a subcellularfraction of a cell line or tissue, enriched for specific cellularorganelles such as nuclei, cytoplasm, plasma membranes, mitochondria,internal membrane structures, Golgi apparatus, endoplasmic reticulum,etc. or specific tissue organelles such as post-synaptic densities frombrain, islets from pancreas, etc.

As used herein, the term “hydrolyzing agent” refers to any one orcombination of a large number of different enzymes, including but notlimited to trypsin, Lysine-C endopeptidase (LysC), arginine-Cendopeptidase (ArgC), Asp-N, glutamic acid endopeptidase (GluC) andchymotrypsin, V8 protease and the like, as well as chemicals, such ascyanogen bromide. In the subject invention one or a combination ofhydrolyzing agents cleave peptide bonds in a protein or polypeptide, ina sequence-specific manner, generating a predictable collection ofshorter peptides (a “digest”). A portion of the biological samples arecontacted with hydrolyzing agent(s) to form a digest of the biologicalsample. Given that the amino acid sequences of certain polypeptides andproteins in biological samples are often known and that the hydrolyzingagent(s) cuts in a sequence-specific manner, the shorter peptides in thedigest are generally of a predicable amino acid sequence. Preferably,the treatment of a polypeptide with a hydrolyzing agents results inabout 2 to about 20, more preferably about 5 to about 15 and mostpreferably about 10 peptides. If the polypeptide in a biological sampleis a ubiquitinated polypeptide, at least one of the resulting peptidesin the digest will be a ubiquitin remnant peptide. The preferredhydrolyzing agent is a protease, or chemical which cleaves ubiquitinatedproteins in a manner that results in the formation of at least oneubiquitin remnant peptide. Most preferably, the protease is trypsin.

The term “mass spectrometer” means a device capable of detectingspecific molecular species and measuring their accurate masses. The termis meant to include any molecular detector into which a polypeptide orpeptide may be eluted for detection and/or characterization. In thepreferred MS procedure, a sample, e.g., the elution solution, is loadedonto the MS instrument, and undergoes vaporization. The components ofthe sample are ionized by one of a variety of methods (e.g., byelectrospray ionization or “ESI”), which results in the formation ofpositively charged particles (ions). The positive ions are thenaccelerated by a magnetic field. The computation of the mass-to-chargeratio of the particles is based on the details of motion of the ions asthey transit through electromagnetic fields, and detection of the ions.The preferred mass measurement error of a mass spectrometer of theinvention is 10 ppm or less, more preferable is 7 ppm or less; and mostpreferably 5 ppm or less.

Fragment ions in the MS/MS and MS³ spectra are generally highly specificand diagnostic for peptides of interest. In contrast, to prior artmethods, the identification of peptide diagnostic signatures providesfor a way to perform highly selective analysis of a complex proteinmixture, such as a cellular lysate in which there may be greater thanabout 100, about 1000, about 10,000, or even about 100,000 differentkinds of proteins. Thus, while conventional mass spectroscopy would notbe able to distinguish between peptides with different sequences butsimilar m/z ratios (which would tend to co-elute with any labeledstandard being analyzed), the use of peptide fragmentation methods andmultistage mass spectrometry in conjunction with LC methods, provide away to detect and quantify target proteins which are only a smallfraction of a complex mixture (e.g., present in less than 2000 copiesper cell or less than about 0.001% of total cellular protein) throughthese diagnostic signatures.

Test peptides are preferably examined by monitoring of a selectedreaction in the mass spectrometer. This involves using the priorknowledge gained by the characterization of a standard peptide and thenrequiring the mass spectrometer to continuously monitor a specific ionin the MS/MS or MS spectrum for both the peptide of interest and thestandard peptide. After elution, the areas-under-the-curve (AUC) forboth the standard peptide and target peptide peaks may be calculated.The ratio of the two areas provides the absolute quantification that maythen be normalized for the number of cells used in the analysis and theprotein's molecular weight, to provide the precise number of copies ofthe protein per cell.

As used herein the term, “accurate mass” refers to an experimentally ortheoretically determined mass of an ion that is used to determine anelemental formula. For ions containing combinations of the elements C,H, N, O, P, S, and the halogens, with mass less than 200 Unified AtomicMass Units, a measurement about 5 ppm uncertainty is sufficient touniquely determine the elemental composition.

As used herein the term, “predetermined peptide accurate mass” refers tothe experimentally determined or calculated accurate mass of a peptidewith a known amino acid sequence (along with any associatedpost-translational modifications). The accurate mass of any suchspecific amino acid sequence may be readily calculated by one of skillin the art.

As used herein, “a peptide fragmentation signature” refers to thedistribution of mass-to-charge ratios of fragmented peptide ionsobtained from fragmenting a peptide, for example, by collision induceddisassociation, ECD, LID, PSD, IRNPD, SID, and other fragmentationmethods. A peptide fragmentation signature which is “diagnostic” or a“diagnostic signature” of a target protein or target polypeptide is onewhich is reproducibly observed when a peptide digestion product of atarget protein/polypeptide identical in sequence to the peptide portionof a standard peptide, is fragmented and which differs only from thefragmentation pattern of the standard peptide by the mass of themass-altering label and/or the presence of a ubiquitin remnant.Preferably, a diagnostic signature is unique to the target protein(i.e., the specificity of the assay is at least about 95%, at leastabout 99%, and preferably, approaches 100%).

The term “substrate” includes any solid support or phase upon which abinding partner may be immobilized. Preferred supports are those wellknown in the art of affinity chromatography for example but not limitedto polymeric and optionally magnetic beads, polystyrene, sepharose oragarose gel matrices, or nitrocellulose membranes.

The term “binding partner” refers to any of a large number of differentmolecules or aggregates. Preferably, a binding partner functions bybinding to a polypeptide or peptide in order to enrich it prior toanalysis, e.g., by MS, LC-MS, or LC-MS/MS. Preferably, binding partnersbind ubiquitin remnant peptides to enrich in a digest. Proteins,polypeptides, peptides, nucleic acids (oligonucleotides andpolynucleotides), antibodies, ligands, polysaccharides, microorganisms,receptors, antibiotics, and test compounds (particularly those producedby combinatorial chemistry) may each be a binding partner.

In the preferred one embodiment, the binding partner is immobilized bybeing directly or indirectly, covalently or non-covalently bound to thesubstrate. In another embodiment, the binding partner does not require asubstrate and can be used to immuno-precipitate the ubiquitin remnantpeptides for example. In a further embodiment, the binding partner canbe used to bind ubiquitin remnant peptides in solution. The techniciancould then enrich for ubiquitin remnant peptides by filtering ubiquitinremnant peptide-binding partner complexes, through size cut-off or sizeexclusion chromatography for example.

The preferred binding partner is a “ubiquitin remnant peptide specificantibody” or an “anti-ubiquitin remnant antibody” which specifically yetreversibly binds ubiquitin remnant peptides and does not bind (i.e.,cross react with) peptides having the same amino acid sequence but whichlack the ubiquitin remnant. As such, the preferred ubiquitin remnantpeptide-specific antibodies bind ubiquitin remnant peptides in a contextindependent manner.

Accordingly, the invention provides an isolated antibody or bindingpartner that preferentially binds a ubiquitin remnant peptide over apeptide having the same amino acid sequence as the ubiquitin remnantpeptide but lacks the ubiquitin remnant. In some embodiments, theisolated antibody or binding partner specifically binds a ubiquitinremnant peptide but does not specifically bind a peptide having the sameamino acid sequence as the ubiquitin remnant peptide but lacks theubiquitin remnant. As used herein, by “specifically binds” is meant thata binding partner or an antibody of the invention interacts with itstarget molecule (e.g., a ubiquitin remnant peptide), where theinteraction is dependent upon the presence of a particular structure(e.g., the antigenic determinant or epitope on the peptide); in otherwords, the reagent is recognizing and binding to a specific polypeptidestructure rather than to all polypeptides in general. In someembodiments, the isolated antibodies or isolated binding partners do notspecifically bind to a peptide having the same amino acid sequence asthe ubiquitin remnant peptide but lacks the ubiquitin remnant.

The isolated antibodies and/or isolated binding partners of theinvention can be used in the methods of the invention.

It should be understood that the substrate can have a number manydifferent binding partners having a different binding specificity for adifferent polypeptide, peptide, subiquitin remnant peptide or epitopesthereof. As such, binding partners might be derived from monoclonalsources or polyclonal sera. Preferably, the substrate has about 2 toabout 500, more preferably about 5 to about 400, even more preferablyabout 10 to about 300 and most preferably about 15 to about 200, yeteven more preferably about 20 to about 100, about 25 to about 75 andabout 30 to about 60 different binding partners each specificallybinding to a different and/or distinct peptide. This allows thetechnician to simultaneously process and analyze the biological samplefor the presence of a large number of polypeptides in a manner notfeasible with multiplex PCR or ELISA techniques. Additional methods andreagents for immunoaffinity purification and/or enrichment of peptidescontaining certain motifs such as the ubiquitin remnant may be found ine.g., in U.S. Pat. Nos. 7,198,896 and 7,300,753.

The term “antibody” as used herein, refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsto an antigen. As such, the term antibody encompasses not only wholeantibody molecules, but also antibody multimers and antibody fragments,as well as variants (including derivatives) of antibodies, antibodymultimers and antibody fragments. The preferred antibody disclosedherein is referred to as D4A7A10.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kilodalton) and one “heavy” chain(about 50-70 kilodalton).

The amino-terminal portion of each chain includes a variable region ofabout, 80, 85, 90, 95, 100, 105, preferably 100 to 110 or more aminoacids primarily responsible for antigen recognition. Herein the terms“heavy chain” and “light chain” refer to the heavy and light chains ofan antibody unless otherwise specified. The amino acid sequence of theD4A7A10 heavy chain is set forth in SEQ ID NO: 1. The amino acidsequence of the D4A7A10 light chain is set forth in SEQ ID NO: 2.

The carboxy-terminal portion of each chain preferably defines a constantregion primarily responsible for effector function. Human light chainsare classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light (“VL”)/heavy chain (“VH”)pair preferably form the antibody binding site. Thus, an intact IgGantibody has two binding sites. Except in bifunctional or bispecificantibodies, the two binding sites are the same. The chains all exhibitthe same general structure of relatively conserved framework regions(FR) joined by three hyper variable regions, also called complementaritydetermining regions or CDRs. The CDRs from the heavy and the lightchains of each pair are aligned by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987 and 1991)),or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al.,Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547 1553 (1992). In addition, bispecificantibodies may be formed as “diabodies” (Holliger et al. “‘Diabodies’:small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448(1993)) or “Janusins” (Traunecker et al. “Bispecific single chainmolecules (Janusins) target cytotoxic lymphocytes on HIV infected cells”EMBO J. 10:3655-3659 (1991) and Traunecker et al. “Janusin: newmolecular design for bispecific reagents” Int J Cancer Suppl 7:51-52(1992)). Production of bispecific antibodies can be a relatively laborintensive process compared with production of conventional antibodiesand yields and degree ofpurity are generally lower for bispecificantibodies.

Examples of molecules which are described by the term “antibody” hereininclude, but are not limited to: single chain Fvs (sdFvs), Fabfragments, Fab′ fragments, F(ab′)2, disulfide linked Fvs (sdFvs), Fvs,and fragments thereof comprising or alternatively consisting of, eithera VL or a VH domain. The term “single chain Fv” or “scFv” as used hereinrefers to a polypeptide comprising a VL domain of antibody linked to aVH domain of an antibody.

Antibodies of the invention include, but are not limited to, monoclonal,multispecific, human or chimeric antibodies, single chain antibodies,Fab fragments, F(ab′) fragments, anti-idiotypic (anti-Id) antibodies(including, e.g., anti-Id antibodies to antibodies of the invention),intracellularly-made antibodies (i.e., intrabodies), and epitope-bindingfragments of any of the above. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG1, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass ofimmunoglobulin molecule. Preferably, an antibody of the inventioncomprises, or alternatively consists of, a VH domain, VH CDR, VL domain,or VL CDR having an amino acid sequence of any one of the antibodieslisted in Table 1, or a fragment or variant thereof. In a preferredembodiment, the immunoglobulin is an IgG1 isotype. In another preferredembodiment, the immunoglobulin is an IgG4 isotype. Immunoglobulins mayhave both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA,and IgY heavy chains may be paired with a light chain of the kappa orlambda forms. Antibodies of the invention may also include multimericforms of antibodies. For example, antibodies of the invention may takethe form of antibody dimers, trimers, or higher-order multimers ofmonomeric immunoglobulin molecules. Dimers of whole immunoglobulinmolecules or of F(ab′)2 fragments are tetravalent, whereas dimers of Fabfragments or scFv molecules are bivalent. Individual monomers withon anantibody multimer may be identical or different, i.e., they may beheteromeric or homomeric antibody multimers. For example, individualantibodies within a multimer may have the same or different bindingspecificities.

Multimerization of antibodies may be accomplished through naturalaggregation of antibodies or through chemical or recombinant linkingtechniques known in the art. For example, some percentage of purifiedantibody preparations (e.g., purified IgG1 molecules) spontaneously formprotein aggregates containing antibody homodimers, and otherhigher-order antibody multimers. Alternatively, antibody homodimers maybe formed through chemical linkage techniques known in the art. Forexample, heterobifunctional crosslinking agents including, but notlimited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1carboxylate] and SATA [Nsuccinimidyl S-acethylthio-acetate] (available,for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can beused to form antibody multimers. An exemplary protocol for the formationof antibody homodimers is given in Ghetie et al., Proceedings of theNational Academy of Sciences USA (1997) 94:7509-7514, which is herebyincorporated by reference in its entirety. Antibody homodimers can beconverted to Fab′2 homodimers through digestion with pepsin. Another wayto form antibody homodimers is through the use of the autophilic T15peptide described in Zhao and Kohler, The Journal of Immunology (2002)25:396-404, which is hereby incorporated by reference in its entirety.

Alternatively, antibodies can be made to multimerize through recombinantDNA techniques. IgM and IgA naturally form antibody multimers throughthe interaction with the mature J chain polypeptide. Non-IgA or non-IgMmolecules, such as IgG molecules, can be engineered to contain the Jchain interaction domain of IgA or IgM, thereby conferring the abilityto form higher order multimers on the non-IgA or non-IgM molecules.(see, for example, Chintalacharuvu et al., (2001) Clinical Immunology101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94,both of which are hereby incorporated by reference in their entireties.)IgA dimers are naturally secreted into the lumen of mucosa-lined organs.This secretion is mediated through interaction of the J chain with thepolymeric IgA receptor (pIgR) on epithelial cells. If secretion of anIgA form of an antibody (or of an antibody engineered to contain a Jchain interaction domain) is not desired, it can be greatly reduced byexpressing the antibody molecule in association with a mutant J chainthat does not interact well with pIgR (Johansen et al., The Journal ofImmunology (2001) 167:5185-5192 which is hereby incorporated byreference in its entirety). ScFv dimers can also be formed throughrecombinant techniques known in the art; an example of the constructionof scFv dimers is given in Goel et al., (2000) Cancer Research60:6964-6971 which is hereby incorporated by reference in its entirety.Antibody multimers may be purified using any suitable method known inthe art, including, but not limited to, size exclusion chromatography.

Monoclonal and polyclonal context-independent ubiquitin remnant peptideantibodies have been identified. For example, the invention encompassesthe monoclonal and polyclonal antibodies listed in Table 1 and the celllines engineered to express them or capable of expressing them.

Further, the present invention encompasses the polynucleotides encodingthe anti-ubiquitin remnant peptide antibodies or portions thereof.Molecules encoding e.g., VH domains, VH CDRs, VL domains, or VL CDRshaving an amino acid sequence of the corresponding region of theinventive antibodies expressed by a cell that specifically bind toubiquitin remnant peptides but not peptides having the same amino acidsequence but lacking the ubiquitin remnant, or fragments or variantsthereof are also encompassed by the invention, as are nucleic acidmolecules that encode these antibodies and/or molecules. In specificembodiments, the present invention encompasses antibodies, or fragmentsor variants thereof that bind to an epitope that comprises the ubiquitinremnant.

Methods for identifying the complementarity determining regions (CDRs)of an antibody by analyzing the amino acid sequence of the antibody arewell known (see, e.g., Wu, T. T. and Kabat, E. A. (1970) J. Exp. Med.132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Moreaet al., Biophys Chem. 68(1-3):9-16 (October 1997); Morea et al., J Mol.Biol. 275(2):269-94 (Jan. 1998); Chothia et al., Nature 342(6252):877-83(December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64(2007).

As one non-limiting example, the following method can be used toidentify the CDRs of an antibody.

For the CDR-L1, the CDR-L1 is approximately 10-17 amino acid residues inlength. Generally, the start is at approximately residue 24 (the residuebefore the 24^(th) residue is typically a cysteine. The CDR-L1 ends onthe residue before a tryptophan residue. Typically, the sequencecontaining the tryptophan is either Trp-Tyr-Gln, Trp-Leu-GlnTrp-Phe-Gln, or Trp-Tyr-Leu, where the last residue within the CDR-L1domain is the residue before the TRP in all of these sequences.

For the CDR-L2, the CDR-L2 is typically seven residues in length.Generally, the start of the CDR-L2 is approximately sixteen residuesafter the end of CDR-L1 and typically begins on the on the residue afterthe sequences of Ile-Tyr, Val-Tyr, Ile-Lys, or Ile-Phe.

For the CDR-L3, the CDR-L3 is typically 7-11 amino acid residues inlength. Generally, the domain starts approximately 33 residues after theend of the CDR-L2 domain. The residue before the start of the domain isoften a cysteine and the domain ends on the residue before Phe in thesequence Phe-Gly-XXX-Gly (where XXX is the three letter code of anysingle amino acid.

For the CDR-H1, the CDR-H1 domain is typically 10-12 amino acid residuesin length and often starts on approximately residue 26. The domaintypically starts four or five residues after a cysteine residue, andtypically ends on the residue before a Trp (the Trp is often found inone of the following sequences: Trp-Val, Trp-Ile, or Trp-Ala.

For the CDR-H2, the CDR-H2 domain is typically 16 to 19 residues inlength and typically starts 15 residues after the final residue of theCDR-H1 domain. The domain typically ends on the amino acid residuebefore the sequence Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala(which includes, for example, the sequences Lys-Leu-Thr andArg-Ala-Ala).

For the CDR-H3, the CDR-H3 domain is typically 3-25 amino acids inlength and typically starts 33 amino acid residues after the finalresidues of the CDR-H2 domain (which is frequently two amino acidresidues after a cysteine residue, e.g., a cysteine in the sequenceCys-Ala-Arg). The domain ends on the amino acid immediately before theTrp in the sequence Trp-Gly-XXX-Gly (where XXX is the three letter codeof any single amino acid).

The inventive anti-ubiquitin remnant peptide antibodies may be coupledto a detectable label such as an enzyme, a fluorescent label, aluminescent label, or a bioluminescent label.

The present invention also provides anti-ubiquitin remnant peptideantibodies that are coupled to a therapeutic or cytotoxic agent. Thepresent invention also provides anti-PA antibodies which are coupled,directly or indirectly, to a radioactive material.

In further embodiments, the anti-ubiquitin remnant peptide antibodies ofthe invention have a dissociation constant (K_(D)) of 10⁻⁷ M or less fora ubiquitin remnant peptide. In preferred embodiments, theanti-ubiquitin remnant peptide antibodies of the invention have adissociation constant (K_(D)) of 10⁻⁹ M or less for a ubiquitin remnantpeptide.

In further embodiments, antibodies of the invention have an off rate(k_(off)) of 10⁻³/sec or less. In preferred embodiments, antibodies ofthe invention have an off rate (k_(off)) of 10⁻⁴/sec or less. In otherpreferred embodiments, antibodies of the invention have an off rate(k_(off)) of 10⁻⁵/sec or less.

The present invention also provides panels of the anti-ubiquitin remnantpeptide antibodies (including molecules comprising, or alternativelyconsisting of, antibody fragments or variants) wherein the panel memberscorrespond to one, two, three, four, five, ten, fifteen, twenty, or moredifferent the anti-ubiquitin remnant peptide antibodies of the invention(e.g., whole antibodies, Fabs, F(ab′)₂ fragments, Fd fragments,disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, andscFvs). The present invention further provides mixtures of theanti-ubiquitin remnant peptide antibodies wherein the mixturecorresponds to one, two, three, four, five, ten, fifteen, twenty, ormore different the anti-ubiquitin remnant peptide antibodies of theinvention (e.g., whole antibodies, Fabs, F(ab′)₂ fragments, Fdfragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id)antibodies, and scFvs)). The present invention also provides forcompositions comprising, or alternatively consisting of, one, two,three, four, five, ten, fifteen, twenty, or more the anti-ubiquitinremnant peptide antibodies of the present invention (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof). A composition of the invention may comprise, oralternatively consist of, one, two, three, four, five, ten, fifteen,twenty, or more amino acid sequences of one or more of theanti-ubiquitin remnant peptide antibodies or fragments or variantsthereof. Alternatively, a composition of the invention may comprise, oralternatively consist of, nucleic acid molecules encoding one or moreantibodies of the invention.

The present invention also provides for fusion proteins comprising ananti-ubiquitin remnant peptide antibody (including molecules comprising,or alternatively consisting of, antibody fragments or variants thereof)of the invention, and a heterologous polypeptide (i.e., a polypeptideunrelated to an antibody or antibody domain). Nucleic acid moleculesencoding these fusion proteins are also encompassed by the invention. Acomposition of the present invention may comprise, or alternativelyconsist of, one, two, three, four, five, ten, fifteen, twenty or morefusion proteins of the invention.

Alternatively, a composition of the invention may comprise, oralternatively consist of, nucleic acid molecules encoding one, two,three, four, five, ten, fifteen, twenty or more fusion proteins of theinvention.

The term “elution solution” refers to a solution that when brought intocontact with the binding partner, results in the dissociation of thepolypeptide or peptide and preferably the ubiquitin remnant peptide fromthe binding partner into the elution solution. Determining the salt, pHand ionic conditions necessary for such functionality is well with theordinary skill in the art. Preferably, the elution solution is enrichedfor polypeptides and peptides which were bound to the binding partnersrelative to the polypeptides and peptides of the digest. Preferably, theelution solution has about 500 to about 5000, more preferably about 1000to about 2000 different peptides. Most preferably, the elution solutionis enriched for ubiquitin remnant peptides. Preferably, a portion of theelution solution is directly transferred to a mass spectrometer, LC-MSor LC-MS/MS. Alternatively, the elution solution is subject to furthermanipulation e.g., to concentrate the peptides and/or polypeptidescontained therein. Mechanisms for directing solutions from liquidchromatography to mass spectrometers may be found for example in U.S.Pub. No. 20080217254.

The term “vaporizing a portion of the elution solution” means that aportion of the elution solution is preferably transferred to a massspectrometer for vaporization and ionization.

The term “ionizing” refers to atmospheric pressure chemical ionization(APCI), chemical ionization (CI), electron impact (O), electrosprayionization (ESI), fast atom bombardment (FAB), field desorption/fieldionization (FD/FI), matrix assisted laser desorption ionization (MALDI),and thermospray ionization. The preferred method of ionization is ESI astends to minimize the propensity of macromolecules to fragment whenionized.

Preferably in ESI, liquid containing the peptides of interest isdispersed by electrospray into a fine aerosol. Preferred solvents forelectrospray ionization are prepared by mixing water with volatileorganic compounds (e.g. methanol, acetonitrile). To decrease the initialdroplet size, compounds that increase the conductivity (e.g. aceticacid) are preferably added to the solution. Large-flow electrosprays mayprovide additional nebulization by an inert gas such as nitrogen. Theaerosol is sampled into the first vacuum stage of a mass spectrometerthrough a capillary, which can be heated to aid further solventevaporation from the charged droplets. Preferably, the solventevaporates from a charged droplet until it becomes unstable uponreaching its Rayleigh limit. At this point, the droplet preferablydeforms and emits charged jets in a process known as Rayleigh fission.During the fission, the droplet loses a small percentage of its massalong with a relatively large percentage of its charge

As used herein, “ionized molecule” refers to molecules in the elutionsolution that have become charged and are ready to move into theelectric fields that will direct them into the mass analyzer of a massspectrometer. Preferably, the ionized molecules include ionizedpolypeptides, peptides and/or ubiquitin remnant peptides present in theelution solution. Most preferably, the ionized molecules are ubiquitinremnant peptides.

The term “standard peptide” as used herein, refers to a peptide thatis 1) recognized as equivalent to a peptide of interest in the digestgenerated by a hydrolyzing agent, e.g., the ubiquitin remnant peptide,by the appropriate binding partner; and 2) differs from the peptide ofinterest in a manner that can be distinguished by a mass spectrometer,e.g., by way of a mass-altering label. Preferably, the standard peptidehas the same amino acid sequence as the ubiquitin remnant peptide but issynthesized utilizing elemental isotopes. Preferably, those isotopes are¹⁵N, ¹³C, ¹⁸O or ²H. Alternatively, a standard peptide can 1) have thesame amino acid sequence as a ubiquitin remnant peptide yet lack theubiquitin remnant; and 2) differ from the ubiquitin remnant peptide in amanner that can be distinguished by a mass spectrometer, e.g., bylacking the ubiquitin remnant. Exemplary standard peptides are describedin U.S. Pub. No. 20060154318 and 20060148093. One or more standardpeptides may be added to the biological sample before or after treatmentwith a hydrolyzing agent such that it co-elutes with the peptide ofinterest into the elution solution. The standard peptide can be addeddirectly to the elution solution.

One aspect of the invention relates to providing methods for determininga site of ubiquitination in a polypeptide. The method comprisesobtaining a plurality of ubiquitinated polypeptides; digesting theubiquitinated polypeptides with a protease, thereby generating aplurality of test peptides; enriching the plurality of test peptides forubiquitin remnant peptides; and determining the presence of a ubiquitinremnant peptide by mass spectrometry, wherein the presence of theubiquitin remnant peptide allows the technician to determine a site ofubiquitination of the polypeptide. The test peptide being evaluated canbe ionized and/or fragmented prior to the determining step. Preferably,ionizing is performed by electrospray.

In one embodiment of this aspect of the invention, the method fordetermining a site of ubiquitination comprises obtaining a plurality ofubiquitinated polypeptides; digesting the ubiquitinated polypeptideswith a protease; thereby generating a plurality of test peptides; atleast some of which comprise a ubiquitin remnant, enriching theplurality of test peptides for ubiquitin remnant peptides; andidentifying a mass difference between a test peptide and a standardpeptide comprising a known identical amino acid sequence as the testpeptide; the mass difference corresponding to the mass of the ubiquitinremnant, wherein detection of the mass difference indicates a site ofubiquitination in the test peptide.

In another aspect, the methods further comprise the step of mapping asequence of a test peptide comprising a ubiquitin remnant to apolypeptide sequence comprising the same amino acid sequence as the testpeptide, thereby determining the site of ubiquitination in thepolypeptide sequence. In another embodiment, the ubiquitin remnantcomprises GlyGly amino acid residues and has a mass of about 114daltons. The methods can be used to detect one or more sites ofubiquitination in a polypeptide, as well as the amount of ubiquitinationat particular sites in a population of polypeptides.

In a further aspect of the invention, ubiquitination sites areidentified for a plurality of polypeptides in a first cell and in asecond cell and the sites identified in the first cell are compared tothose in the second cell. In one aspect, the first cell is a normal cell(e.g., from a healthy patient), while the second cell is from a patientwith a pathological condition (e.g., a neurodegenerative disease,cancer, a disease of the immune system). Preferably, the second cell isthe target of the pathology (e.g., a tumor cell from a cancer patient; aneural cell from a patient with a neurodegenerative disease). In anotherembodiment of this aspect of the invention, the second cell differs fromthe first cell in expressing one or more recombinant DNA molecules, butis otherwise genetically identical to the first cell. In a furtherembodiment, the site of ubiquitination is correlated with disease anddetection of ubiquitination at the site is associated with risk of thedisease. In another embodiment, the disease is a eurodegenerativedisease, such as Alzheimer's or Pick's disease. In another aspect, thedisease is cancer. In a further aspect, the disease is an abnormalimmune response or inflammatory disease.

In another aspect of the invention, the methods disclosed herein areused to identify regulators of ubiquitination pathways. In oneembodiment, the methods further comprise contacting a first cell with acompound and comparing ubiquitination sites identified in the first cellwith ubiquitination sites in a second cell not contacted with thecompound. The compound may be a therapeutic agent for treating a diseaseassociated with an improper state of ubiquitination (e.g., abnormalsites or amounts of ubiquitination). Suitable agents include, but arenot limited to, drugs, polypeptides, peptides, antibodies, nucleic acids(genes, cDNA's, RNA's, antisense molecules, siRNA/miRNA constructs,ribozymes, aptamers and the like), toxins, and combinations thereof.

Preferably, the methods further comprise generating a databasecomprising data files storing information relating to ubiquitinationsites for a plurality of polypeptides for a plurality of differentcells. Preferably, the data files also include information relating toamount of ubiquitination of a polypeptide in at least one cell.Additionally, the database comprises data relating to the source of thecell (e.g., such as a patient).

The invention further provides a computer memory comprising data filesstoring information relating to ubiquitination sites for a plurality ofpolypeptides for a plurality of different cells.

In another aspect of the invention, substantially purified testpeptides, preferably ubiquitin remnant peptides, obtained after one ormore separation steps are analyzed by a peptide analyzer that evaluatesthe mass of the peptide or a fragment thereof. Suitable peptideanalyzers include, but are not limited to, a mass spectrometer, massspectrograph, single-focusing mass spectrometer, static field massspectrometer, dynamic field mass spectrometer, electrostatic analyzer,magnetic analyzer, quadropole analyzer, time of flight analyzer (e.g., aMALDI Quadropole time-of-flight mass spectrometer), Wien analyzer, massresonant analyzer, double-focusing analyzer, ion cyclotron resonanceanalyzer, ion trap analyzer, tandem mass spectrometer, liquid secondaryionization MS, and combinations thereof in any order (e.g., as in amulti-analyzer system). Such analyzers are known in the art and aredescribed in, for example, Mass Spectrometry for the BiologicalSciences, Burlingame and Can eds., Human Press, Totowa, N.J.)

In general, any analyzer can be used that can separate matter accordingto its anatomic and molecular mass. Preferably, the peptide analyzer isa tandem MS system (an MS/MS system) since the speed of an MS/MS systemenables rapid analysis of low femtomole levels ofpeptide and can be usedto maximize throughput.

In a preferred embodiment of this aspect of the invention, the peptideanalyzer comprises an ionizing source for generating ions of a testpeptide and a detector for detecting the ions generated. The peptideanalyzer further comprises a data system for analyzing mass datarelating to the ions generated and for deriving mass data relating tothe test peptide.

A sample comprising a test peptide can be delivered to the peptideanalyzer using a delivery mechanism as described above. Interfacesbetween a sample source (e.g., an HPLC column) and ion source can bedirect or indirect. For example, there may be an interface that providesfor continuous introduction of the sample to the ion source.Alternatively, sample can be intermittently introduced to the ion source(e.g., in response to feedback from the system processor during theseparation process, or while the separation system is off-line).

In another embodiment, the ion source is an electrospray which is usedto provide droplets to the peptide analyzer, each droplet comprising asubstantially purified test peptide obtained from previous separationstep(s) (e.g., such as HPLC or reversed phase liquid chromatography).During electrospray, a high voltage is applied to a liquid streamcausing large droplets to be subdivided into smaller and smallerdroplets until a peptide enters the gas phase as an ion. Ionizationgenerally is accomplished when the test peptide loses or gains a protonat one or more sites on the peptide (e.g., at the amino terminus, and/orat lysine and arginine residues). Ionization in electrospray isconstant; MALDI can be used to achieve pulsed ionization. Other methodsof ionization, include but are not limited to, plasma desorptionionization, thermospray ionization, and fast atom bombardment ionizationas are known in the art.

When MALDI is used, peptides can be delivered to a solid support, e.g.,sample plate inserted into the mass spectrometer. The support maycomprise a light-absorbent matrix. In another embodiment, asubstantially purified ubiquitinated polypeptide is provided on a sampleplate and protease digestion occurs on the sample plate prior toionization. For example, substantially purified ubiquitinated peptidesalso can be obtained from protease digests as described above andseparated by a liquid chromatography method. Preferably, the peptideanalyzer further comprises an ion transfer section through which ionsare delivered from the ion source to the detector. The ion transfersection comprises an electric and/or magnetic field generator (e.g., anelectrode ring) that modulates the acceleration of ions generated by theionizing source. The electric/magnetic field generator directs ionsthrough the ion transfer section of the peptide analyzer to the iondetector.

Preferably, the peptide analyzer further comprises an ion trappositioned between the ion transfer section of the analyzer and thedetector, for performing one or more operations such as ion storage, ionselection and ion collision. The ion trap can be used to fragment ionsproduced by the ion source (e.g., causing ions to undergo collisionalactivated dissociation in the presence of a neutral gas ions, such ashelium ions). The ion trap also can be used to store ions in stableorbits and to sequentially eject ions based on their mass-to-chargevalues (m/z) to the detector. An additional separation section can beprovided between the ion trap and detector to separate fragmentsgenerated in the ion trap (e.g., as in tandem MS). The detector detectsthe signal strength of each ion (e.g., intensity), which is a reflectionof the amount of protonation of the ion.

The peptide analyzer additionally preferably is associated with datasystem for recording and processing information collected by thedetector. The data system can respond to instructions from a processorin communication with the separation system and also can provide data tothe processor. Preferably, the data system includes one or more of acomputer; an analog to digital conversion module; and control devicesfor data acquisition, recording, storage and manipulation. Morepreferably, the device further comprises a mechanism for data reduction,i.e., a device to transform the initial digital or analog representationof output from the analyzer into a form that is suitable forinterpretation, such as a graphical display, a table of masses, a reportof abundances of ions, etc.)

The data system can perform various operations such as signalconditioning (e.g.,

providing instructions to the peptide analyzer to vary voltage, current,and other operating parameters of the peptide analyzer), signalprocessing, and the like. Data acquisition can be obtained in real time,e.g., at the same time mass data is being generated. However, dataacquisition also can be performed after an experiment, e.g., when themass spectrometer is off line.

The data system can be used to derive a spectrum graph in which relativeintensity (i.e., reflecting the amount of protonation of the ion) isplotted against the mass to charge ratio (m/z ratio) of the ion or ionfragment. An average of peaks in a spectrum can be used to obtain themass of the ion (e.g., peptide) (see, e.g., McLafferty and Turecek,1993, Interpretation of Mass Spectra, University Science Books, CA).

Mass spectra can be searched against a database of reference peptides ofknown mass

and sequence to identify a reference peptide which matches a testpeptide (e.g., comprises a mass which is smaller by the amount of massattributable to a ubiquitin remnant). The database of standard peptidescan be generated experimentally, e.g., digesting non-ubiquitinatedpeptides and analyzing these in the peptide analyzer. The database alsocan be generated after a virtual digestion process, in which thepredicted mass of peptides is generated using a suite of programs suchas PROWL (e.g., available from ProteoMetrics, LLC, New York; N.Y.). Anumber of database search programs exist which can be used to correlatemass spectra of test peptides with amino acid sequences from polypeptideand nucleotide databases, including, but not limited to: the SEQUESTprogram (Eng, et al., J. Am. Soc. Mass Spectrum. 5: 976-89; U.S. Pat.No. 5,538,897; Yates, Jr., III, et al., 1996, J. Anal. Chem. 68(17):534-540A), available from Finnegan Corp., San Jose, Calif.

Data obtained from fragmented peptides can be mapped to a larger peptideor polypeptide sequence by comparing overlapping fragments. Preferably,a Ubiquitinated peptide is mapped to the larger polypeptide from whichit is derived to identify the ubiquitination site on the polypeptide.Sequence data relating to the larger polypeptide can be obtained fromdatabases known in the art, such as the nonredundant protein databasecompiled at the Frederick Biomedical Supercomputing Center at Frederick,Md.

In another aspect of the invention, the amount and location ofubiquitination is compared to the presence, absence and/or quantity ofother types ofpolypeptide modifications. For example, the presence,absence, and/or quantity of phosphorylation, sulfation, glycosylation,and/or acetylation can be determined using methods routine in the art(see, e.g., Rossomando, et al., 1992, Proc. Natl. Acad. Sci. USA 89:5779-578; Knight et al., 1993, Biochemistry 32: 2031-2035; U.S. Pat. No.6,271,037). The amount and locations of one or more modifications can becorrelated with the amount and locations of ubiquitination sites.Preferably, such a determination is made for multiple cell states.

Knowledge of ubiquitination sites can be used to identify compounds thatmodulate particular ubiquitinated polypeptides (either preventing orenhancing ubiquitination, as appropriate, to normalize theubiquitination state of the polypeptide). Thus, in one aspect, themethod described above may further comprise contacting a first cell witha compound and comparing ubiquitination sites/amounts identified in thefirst cell with ubiquitination sites/amounts in a second cell notcontacted with the compound. Suitable cells that may be tested include,but are not limited to: neurons, cancer cells, immune cells (e.g., Tcells), stem cells (embryonic and adult), undifferentiated cells,pluripotent cells, and the like. In one preferred aspect, patterns ofubiquitination are observed in cultured cells, such as P 19 cells,pluripotent embryonic carcinoma cells capable of differentiating intocardiac cells and skeletal myocytes upon exposure to DMSO (see Montross,et al., J. Cell Sci. 113 (Pt. 10): 1759-70).

Compounds which can be evaluated include, but are not limited to: drugs;toxins; proteins; polypeptides; peptides; amino acids; antigens; cells,cell nuclei, organelles, portions of cell membranes; viruses; receptors;modulators of receptors (e.g., agonists, antagonists, and the like);enzymes; enzyme modulators (e.g., such as inhibitors, cofactors, and thelike); enzyme substrates; hormones; nucleic acids (e.g., such asoligonucleotides; polynucleotides; genes, cDNAs; RNA; antisensemolecules, ribozymes, aptamers); and combinations thereof. Compoundsalso can be obtained from synthetic libraries from drug companies andother commercially available sources known in the art (e.g., including,but not limited to the LeadQuest® library) or can be generated throughcombinatorial synthesis using methods well known in the art. A compoundis identified as a modulating agent if it alters the site ofubiquitination of a polypeptide and/or if it alters the amount ofubiquitination by an amount that is significantly different from theamount observed in a control cell (e.g., not treated with compound).

In further aspect of the invention, the ubiquitination states (e.g.,sites and amount of ubiquitination) of first and second cells areevaluated. Preferably, the second cell differs from the first cell inexpressing one or more recombinant DNA molecules, but is otherwisegenetically identical to the first cell. Alternatively, or additionally,the second cell can comprise mutations or variant allelic forms of oneor more genes. In one aspect, DNA molecules encoding regulators of theubiquitin pathway can be introduced into the second cell (e.g., E1, E2,E3, deubiquitinating proteins, fragments thereof, mutant forms thereof,variants, and modified forms thereof, or compounds identified as above)and alterations in the ubiquitination state in the second cell can bedetermined. DNA molecules can be introduced into the cell using methodsroutine in the art, including, but not limited to: transfection,transformation, electroporation, electro fusion, microinjection, andgermline transfer.

The invention also provides methods for generating a database comprisingdata files for storing information relating to diagnostic peptidefragmentation signatures. Preferably, data in the data files include oneor more peptide fragmentation signatures characteristic or diagnostic ofa cell state (e.g., such as a state which is characteristic of adisease, a normal physiological response, a developmental process,exposure to a therapeutic agent, exposure to a toxic agent or apotentially toxic agent, and/or exposure to a condition). Data in thedata files also preferably includes values corresponding to level ofproteins corresponding to the peptide fragmentation signatures found ina particular cell state.

In one embodiment, for a cell state determined by the differentialexpression of at least one protein, a data file corresponding to thecell state will minimally comprise data relating to the mass spectraobserved after peptide fragmentation of a standard peptide diagnostic ofthe protein. Preferably, the data file will include a valuecorresponding to

the level of the protein in a cell having the cell state. For example, atumor cell state is associated with the overexpression of p53 (see,e.g., Kern, et al., 2001, Int. J. Oncol. 21(2): 243-9). The data filewill comprise mass spectral data observed after fragmentation of astandard corresponding to a subsequence of p53. Preferably, the datafile also comprises a value relating to the level of p53 in a tumorcell. The value may be expressed as a relative value (e.g., a ratio ofthe level of p53 in the tumor cell to the level of p53 in a normal cell)or as an absolute value (e.g., expressed in nM or as a % of totalcellular proteins).

Preferably, the data files also include information relating to thepresence or amount of a modified form of a target a polypeptide in atleast one cell and to mass spectral data diagnostic of the modified form(i.e., peak data for a fragmented peptide internal standard whichcorresponds to the modified form). More preferably, the data files alsocomprise spectral data diagnostic of the unmodified form as well as datacorresponding to the level of the unmodified form.

In one embodiment, data relating to ubiquitination sites and amounts ofubiquitination are stored in a database to create a proteome map ofubiquitinated proteins. Preferably, the database comprises a collectionof data files relating to all ubiquitinated polypeptides in a particularcell type. The database preferably further comprises data relating tothe origin of the cell, e.g., such as data relating to a patient fromwhom a cell was obtained. More preferably, the database comprises datarelating to cells obtained from a plurality of patients. In one aspect,the database comprises data relating to the ubiquitination of aplurality of different cell types (e.g., cells from patients with apathology, normal patients, cells at various stages of differentiation,and the like). In another aspect, data relating to ubiquitinationpatterns in cells obtained from patients with a neurological disease arestored in the database. For example, information relating toubiquitination in cell samples from patients having any of Alzheimer'sdisease; amyotrophic lateral sclerosis; dementia; depression; Down'ssyndrome; Huntington's disease; peripheral neuropathy; multiplesclerosis; neurofibromatosis; Parkinson's disease; and schizophrenia,can be included in the database.

In a further embodiment, data relating to ubiquitination patterns incells from patients with cancer are stored in the database, including,but not limited to patients with: adenocarcinoma; leukemia; lymphoma;melanoma; myeloma; sarcoma; teratocarcinoma; and, in particular, cancersof the adrenal gland; bladder; bone; bone marrow; brain; breast; cervix;gall bladder; ganglia; gastrointestinal; tract; heart, kidney; liver;lung; muscle; ovary; pancreas; parathyroid; prostate; salivary glands;skin; spleen; testes; thymus; thyroid; and uterus.

Additionally, data of ubiquitination patterns in cells from patientswith an immune disorder may be included in the database. Such a disordercan include: acquired immunodeficiency syndrome (AIDS); Addison'sdisease; adult respiratory distress

syndrome; allergies; ankylosing spondylitis; amyloidosis; anemia;asthma; atherosclerosis; autoimmune hemolytic anemia; autoimmunethyroiditis; bronchitis; cholecystitis; contact dermatitis; Crohn'sdisease; atopic dermatitis; dermatomyositis; diabetes mellitus;emphysema; episodic lymphopenia with lymphocytotoxins; erythroblastosisfetalis; erythema nodosum; atrophic gastritis; glomerulonephritis;Goodpasture's syndrome; gout; Graves' disease; Hashimoto's thyroiditis;hypereosinophilia; irritable bowel syndrome; myasthenia gravis;myocardial or pericardial inflammation; osteoarthritis; osteoporosis;pancreatitis; polymyositis; psoriasis; Reiter's syndrome; rheumatoidarthritis; scleroderma; Sjogren's syndrome; systemic anaphylaxis;systemic lupus erythematosus; systemic sclerosis; thrombocytopenicpurpura; ulcerative colitis; uveitis; Werner syndrome; and viral,bacterial, fungal, parasitic, protozoal, and helminthic infections.

Data regarding ubiquitination in apoptotic cells and in pathologiesassociated with the misregulation of apoptosis also can be obtainedusing methods according to the invention.

In a further embodiment, data regarding ubiquitination in cardiac cellsand cells from patients exhibiting a cardiac disease or at risk for acardiac disease are obtained. In one aspect, the disease is aninfarction or a condition relating to ischemia. In another aspect, thedisease is cardiomyopathy.

Another aspect of the invention provides for kits for detecting and/orquantifying a polypeptide modification, such as ubiquitination. In oneembodiment, the kit comprises a ubiquitin remnant specific bindingpartner and one or more components, including, but not limited to: aprotease, preferably trypsin; a ubiquitinated molecule comprising knownubiquitination sites; acetonitrile; silica resin; heptafluorobutyricacid; urea (e.g., 8M urea); a sample plate for use with a massspectrometer; a light-absorbent matrix; an ion exchange resin; softwarefor analyzing mass spectra (e.g., such as SEQUEST); fused silicacapillary tubing; and access to a computer memory comprising data filesstoring information relating to ubiquitination sites for a plurality ofpolypeptides for a plurality of different cells. Access may be in theform of a computer readable program product comprising the memory, or inthe form of a URL and/or password for accessing an internet site forconnecting a user to such a memory.

EXAMPLES Example 1

Both polyclonal and monoclonal antibodies capable of recognizing theremnant of ubiquitin left from ubiquitinated proteins after digestionwith the protease trypsin were generated. These antibodies weregenerated using a synthetic peptide library immunogen with the sequenceCXXXXXXK(GG)XXXXXX, i.e., a Cysteine residue at the peptideamino-terminus, 6 “X” residues (X=any amino acid selected from allcommon amino acids excluding cysteine and tryptophan), a lysine residue(“K”) that has been modified by addition of a Glycine-Glycine dipeptideto the epsilon-amino group of that lysine residue and 6 more “X”residues.

Polyclonal antibodies were generated by injecting rabbits with thepeptide library immunogen described above conjugated either to keyholelimpet hemocyanin (KLH) or blue carrier protein. K(GG)-specificpolyclonal antibodies from 6 rabbits: BL3415, BL3416, BL4933, BL4934,BL4935, BL4936.

BL4933, BL4935 were used as starting material for monoclonal antibodydevelopment.

A monoclonal antibody from BL4933 was cloned and named recombinantantibody #3925 (D4A7A10). An additional monoclonal antibody was clonedfrom BL4935 (D24B6G9).

Table 1 shows the different monoclonal and polyclonal anti-ubiquitinremnant antibodies of the invention.

Monoclonal anti-Ubiquitin Polyclonal anti-Ubiquitin Remnant AntibodiesRemnant Antibodies BL3415 BL3416 D4A7A10 BL4933 BL4934 D28B6G9 BL4935BL4936

The heavy chain amino acid sequence of the D4A7A10 clone is provided inSEQ ID NO: 1. The light chain amino acid sequence of the D4A7A10 cloneis provided in SEQ ID NO: 2. For the D4A7A10 clone (i.e., antibody#3925), using the CDR-defining rules set forth above, the CDR regionsfor the heavy and light chain are as follows:

Heavy Chain: CDR1 (SEQ ID NO: 3) GFTISSNYYIYWV CDR2 (SEQ ID NO: 4)CIYGGSSGTTLYASWAKG CDR3 (SEQ ID NO: 5) DFRGADYSSYDRIWDTRLDL Light Chain:CDR1 (SEQ ID NO: 6) QSSENVYNKNWLS CDR2 (SEQ ID NOL: 7) KASTLAS CDR3 (SEQID NO: 8) AGDYGGTGDAFV

The skilled artisan can readily determine the CDRs for the otherantibodies disclosed herein including, without limitation, the antibodyD24B6G9 cloned from BL4935.

Example 2

Characterization and Screening of Ubiquitin Tag Motif Antibodies.Anti-ubiquitin remnant peptide antibodies were characterized bydifferential peptide ELISA against antigen peptides CXXXXXXK(GG)XXXXXX(C02-1257) and control peptides CXXXXXXKXXXXXX (173-92A). All antibodiesgave strong positive signals with antigen peptides and showed no bindingwith control peptides. Antibodies were validated by the peptideimmunoprecipitation-MS methods described below by identifyingubiquitin-modified peptides in a trypsin-digested Jurkat cell lysate:antibodies passed this validation test when their use resulted inidentification of most of the seven known ubiquitination sites inubiquitin itself. These seven sites are shown in Table 2. Note that thesome of the sites are represented in more than one peptide produced bytrypsin digestion due to more than one trypsin cleavage sequence nearthe ubiquitinated site and/or due to more than one ubiquitinatablelysine residue in the peptide. For example, the ubiquitinated site atresidue 48 is found in three trypic peptides (see Table 2).

TABLE 2 Known Ubiquitination Sites in Ubiquitin (where the asteriskfollowing the lysing residue (i.e., K*) indicates the ubiquitinatedresidue) Residue Number Peptide Sequences 6 MQIFVK*TLTGK (SEQ ID NO: 9)11 TLTGK*TITLEVEPSDTIENVK (SEQ ID NO: 10) TLTGK*TITLEVEPSDTIENVKAK (SEQID NO: 27 TITLEVEPSDTIENVK*AKIQDKEGIPPDQQR (SEQ ID NO: 

29 AK*IQDKEGIPPDQQR (SEQ ID NO: 13) AK*IQDK*EGIPPDQQR (SEQ ID 33IQDK*EGIPPDQQR (SEQ ID NO: 15) AKIQDK*EGIPPDQQR (SEQ ID NO: 16)AK*IQDK*EGIPPDQQR (SEQ ID 48 LIFAGK*QLEDGR (SEQ ID NO: 18)LIFAGK*QLEDGRTLSDYNIQK (SEQ ID NO: 19) LIFAGK*QLEDGRTLSDYNIQKESTLHLVLR(SEQ ID NO: 63 TLSDYNIQK*ESTLHLVLR (SEQ ID NO: 21)

indicates data missing or illegible when filed

The antibodies of the invention were designed to recognize any peptidethat contains ubiquitinated lysine residues regardless of surroundingpeptide sequences. To illustrate the general context-independentrecognition properties of one of these antibodies, the heat map shown inFIG. 2 shows the frequency of amino acids found with the BL4936polyclonal antibody in a study of four mouse tissues. The studies weresimilar to the study described below in Example 3. Briefly, and by wayof example, the cellular proteins are isolated from the tissue anddigested with trypsin protease. Peptide purification was carried out,e.g., using Sep-PakC18 columns as described in Rush et al., U.S. Pat.No. 7,300,753). Following purification, peptides are lyophilized andthen resuspended in MOPS buffer (50 mM MOPS/NaOH pH 7.2, 10 mM Na₂HPO₄,50 mM NaC1) and insoluble material removed by centrifugation at 12,000×gfor 10 minutes. The anti-ubiquitin remnant antibodies of the inventionwere coupled non-covalently to protein G agarose beads (Roche) at 4mg/ml beads overnight at 4° C. After coupling, antibody-resin was washedtwice with PBS and three times with MOPS buffer. Immobilized antibody(40 1[11, 160 iug) was added as a 1:1 slurry in MOPS IP buffer to thesolubilized peptide fraction, and the mixture was incubated overnight at4° C. The immobilized antibody beads were washed three times with MOPSbuffer and twice with ddH20. Peptides were eluted twice from beads byincubation with 50 IA of 0.15% TFA for 15 minutes each, and thefractions were combined and analyzed by LC-MS/MS mass spectrometry.

Altogether 1458 non-redundant peptides were included in the frequencymap shown in FIG. 2. The map clearly shows there are no stronglypreferred amino acids at least seven residues to the amino-terminal sideof K(GG) modification sites (−7 to −1 in FIG. 2) or at least sevenresidues to the carboxyl-terminal side of K(GG) modification sites (1 to7 in FIG. 2).

Example 3

Numerous experiments were performed using the isolated antibodies of theinvention in the methods described in U.S. Pat. Nos. 7,198,896 and7,300,753. Table 3 lists some of these experiments performed and thenumber ofubiquitinated peptides observed (both redundant andnon-redundant) in each of these experiments.

TABLE 3 Expt Antibody Cell/Tissue Type Treatment 1 Treatment 2 RedundantNon-Redundant 3114 BL4936 mouse heart 447 332 3115 BL4936 mouse liver790 591 3116 BL4936 Embryo mouse 662 548 3117 BL4936 Adult mouse brain735 565 3573 BL4936 rat brain sham mock surgery 738 553 3574 BL4936 ratbrain sham mock surgery 833 618 3575 BL4936 rat brain ischemiaReperfusion 760 554 ischemia 30 R 3576 BL4936 rat brain ischemiaReperfusion 809 580 ischemia 30′ R 3577 BL4936 rat brain ischemiaReperfusion 741 551 ischemia 24 h R 3578 BL4936 rat brain ischemiaReperfusion 773 567 ischemia 24 h R 3970 BL4936 rat brain sham untreated693 499 3971 BL4936 rat brain Reperfusion 829 604 ischemia 30′ R 3972BL4936 rat brain Reperfusion 816 620 ischemia 24 h R 4120 BL4934 ADcontrol untreated 413 271 4121 BL4934 AD control untreated 382 249 4122BL4934 AD control untreated 388 265 4123 BL4934 AD control untreated 488326 4124 BL4934 AD control untreated 406 278 4125 BL4934 AD controluntreated 478 321 4126 BL4934 AD+/− untreated 453 324 4127 BL4934 AD+/−untreated 508 343 4128 BL4934 AD+/− untreated 384 258 4129 BL4934 AD+/−untreated 265 181 5338 BL4934 Jurkat pervanadate calyculin 217 173 5339BL4936 Jurkat pervanadate calyculin 202 161 5566 BL4934 MKN-45 Su11274668 394 5567 BL4934 MKN-45 Su11274 565 353 5642 BL4933 H2228 silac1 DMSO615 408 5643 BL4933 H2228 silac2 inhibitor 556 326 5644 BL4933 H2228silac3 inhibitor 463 298 5645 BL4933 H2228 silac4 inhibitor 415 272 5712D24B6G Jurkat pervanadate calyculin 137 105 5972 BL49 H3122 Silacinhibitor 353 200 5973 BL49 H3122 Silac inhibitor 247 185 5974 BL49H3122 Silac inhibitor 391 245 6090 BL49 H2228 silac inhibitor 193 135Dana Farber 6093 BL49 H3122 Silac inhibitor 178 140 Dana Farber 6131BL49 H1703 normal 978 691 6362 D24B Jurkat pervanad calyculin 431 2836586 BL49 U266 control 793 539 6587 BL49 U266 MG132 791 522 6588 BL49U266 MG132 1074 867 6589 BL49 H929 control 1265 764 6590 BL49 H929 MG132712 468 6591 BL49 H929 MG132 551 467 6846 BL49 H1703 735 484 6847 BL49H1703 1143 841 6916 BL49 RAW 264.7 normal 1366 736 6917 BL49 RAW 264.7LPS 1396 746 6918 BL49 RAW 264.7 LPS 1424 771 6919 BL49 RAW 264.7 MG1321473 871 6939 D4A7 Jurkat pervanad calyculin 286 240 6941 BL49 Jurkatpervanad calyculin 130 102 8149 D4A7 Jurkat calyculin pervanad 613 4458158 D4A7 mouse muscle untreated 886 651 8159 D4A7 mouse spleenuntreated 1355 1033 8160 D4A7 mouse testis untreated 1096 872 8161 D4A7mouse thymus untreated 827 623 8241 D4A7 LNCaP control 940 801 8242 D4A7LNCaP AAG 978 826 8243 D4A7 LNCaP AAG 561 474 8244 D4A7 LNCaPGeldanamycin 874 747 8245 D4A7 LNCaP Geldanamycin 665 569 8246 D4A7LNCaP Velcade 970 8247 D4A7 LNCaP Velcade 1056

The exemplary results shown below in Table 4 correspond to experimentnumber 3115 in Table 3. In the experiment, cellular proteins from 500 mgof mouse liver were denatured with urea, reduced with dithiothreitol,alkylated with iodoacetamide digested with the protease trypsin. Theresulting peptides were separated from other cellular materials byreversed-phased solid phase extraction, then lyophilized andresuspended. Peptides containing the K(GG) modification were separatedfrom other peptides by treatment with the immobilized polyclonalanti-ubiquitin remnant antibody BL4936. The BL4936 associated beads werewashed, and bound K(GG)-peptides were eluted with dilutetrifluouroacetic acid. The peptides were concentrated and then analyzedby liquid chromatography-tandem mass spectrometry LC-MS. See forexample, U.S. Pat. Nos. 7,198,896 and 7,300,753, the entire disclosuresof which are incorporated by reference.

TABLE 4 Known and Novel Ubiquitination Sites Found in One Analysis ofProteins 15 from Mouse Liver SEQ Ubiquitinated ID Row Protein TypeProtein Residue Peptide Sequence NO: 1 Unassigned ADRM1 %34MSLK*GTTVTPDKRK 22 2 Unassigned ADRM1 %34 MSLK*GTTVTPDKR 23 3 UnassignedADRM1 %34 MSLK*GTTVTPDK 24 4 Unassigned ADRM1 %34 M#SLK*GTTVTPDKR 25 5Unassigned ADRM1 %34 M#SLK*GTTVTPDKRK 26 6 Receptor, GLT1 %517MQEDIEMTK*TQSIYDDKN 27 channel, HR transporter or cell surface protein 7Chromatin, HID; H1C %45; %46 KASGPPVSELITK*AVAASK 28 DNA- binding, DNArepair or DNA replication protein 8 Chromatin, H1D; H1E; %63;K*ALAAAGYDVEK 29 DNA- H1C; H1T %63; binding, %64; %66 DNA repair or DNAreplication protein 9 Chromatin, H1D; H1E; %63; K*ALAAAGYDVEKNNSR 30DNA- H1C; H1T %63; binding, %64; %66 DNA repair or DNA replicationprotein 10 Chromatin, H1D; H1E; %74; %74; ALAAAGYDVEK*NNSR 31 DNA- H1C;H1T %75; %77 binding, DNA repair or DNA replication protein 11Chromatin, H1E %45 KTSGPPVSELITK*AVAASK 32 DNA- binding, DNA repair orDNA replication protein 12 Chromatin, H1E %45 TSGPPVSELITK*AVAASK 33DNA- binding, DNA repair or DNA replication protein 13 Chromatin, H2A.1;%119; VTIAQGGVLPNIQAVLLPK* 34 DNA- H2A0; H2AE %118; KTESHH K binding,%119 DNA repair or DNA replication protein 14 Chromatin, H2A.1; %120;119; VTIAQGGVLPNIQAVLLPKK 35 DNA- H2A0; H2AE %120 *TESHH K binding, DNArepair or DNA replication protein 15 Chromatin, H2A.1; %119;VTIAQGGVLPNIQAVLLPK* K 36 DNA-binding, H2AX; %118; DNA repair orHIST2H2AB; 19; DNA HIST2H2AC; 119; %118; replication H2A0; %119; proteinH2A.4; %119; H2AE; %119; H2AL; %119 H2AFJ 16 Chromatin, H2A.1; %120;VTIAQGGVLPNIQAVLLPKK * 37 DNA- H2AX; %119; 120; binding, HIST2H2AB; 120;DNA repair HIST2H2AC; 119; 120; or DNA H2A0; %120; replication H2A.4;%120; protein H2AE; %120 H2AL; H2AFJ 17 Unassigned H2AE %120VTIAQGGVLPNIQAVLLPKK 38 *TESHH KPK 18 Chromatin, H2AFJ %119VTIAQGGVLPNIQAVLLPK* 39 DNA- KTESQK binding, DNA repair or DNAreplication protein 19 Chromatin, H2AFJ %120 VTIAQGGVLPNIQAVLLPKK 40DNA- binding, DNA repair or DNA 20 Chromatin, H2AFY %116GVTIASGGVLPNIHPELLAK* 41 DNA- binding, DNA repair or DNA 21 Chromatin,H2AFY %116 GVTIASGGVLPNIHPELLAK* 42 DNA- binding, DNA repair or DNA 22Chromatin, H2AFY %117 GVTIASGGVLPNIHPELLAKK 3 DNA- *R binding, DNArepair or DNA replication protein 23 Chromatin, H2AL %120VTIAQGGVLPNIQAVLLPKK 4 DNA- *TETHH K binding, DNA repair or DNAreplication protein 24 Chromatin, H2AX %119 K*SSATVGPK 5 DNA- binding,DNA repair or DNA replication protein 25 Chromatin, H2AX; %118; 119LLGGVTIAQGGVLPNIQAVL 46 DNA- HIST2H2AB LPK*K binding, DNA repair or DNAreplication protein 26 Chromatin, H2AX; %118; 119 NDEELNKLLGGVTIAQGGV 47DNA- HIST2H2AB LPNIQA binding, VLLPK*K DNA repair or DNA replicationprotein 27 Chromatin, H2B; %120; AVTK*YTSSK 48 DNA-binding, H2B1D; %120;DNA repair or H2B1A; %122; DNA H2B1N; %121; replication H2B2E; %121;protein H2B1H; %121; H2B1C; %121; Hist3h2ba %121 28 Chromatin, H2B; %46;%46; VLK*QVHPDTGISSK 49 DNA- H2B1D; %48; %47; binding, H2B1A; %47; %47;DNA repair or H2B1N; %47; %47; DNA H2B2E; %47 replication H2B1L; proteinH2B1H; H2B1C; Hist3h2ba 29 Chromatin, H2B; %116; HAVSEGTK*AVTK 50DNA-binding, H2B1D; %116; DNA repair or H2B1A; %118; DNA H2B1N; %117;replication H2B2E; %117; protein H2B1L; %117; H2B1H; %117; H2B1C; %117;Hist3h2ba %117 30 Chromatin, H2B1L %121 AVTK*YTSAK 51 DNA- binding, DNArepair or DNA replication protein 31 Unassigned HIST2H2AB; 119; 119;VTIAQGGVLPNIQAVLLPK* 52 HIST2H2AC; %119 KTESHK H2A.4 32 UnassignedHIST2H2AB; 125; 125; VTIAQGGVLPNIQAVLLPKK 53 HIST2H2AC; %125 TESHK*H2A.4 33 Chaperone HSC70; 507; ITITNDK*GR 54 HSPA1L; 509; HSPA2; 510;HSP70-2; 507; HSP70 %507 34 Ubiquitin NEDD8 %48 LlYSGK*QMNDEK 55conjugating system 35 Unassigned RPS20 %8 DTGK*TPVEPEVAIHR 56 36 UnknownSPG20 %360 SSHPSEPPK*EASGTDVR 57 function 37 Protein Titin %30428EAFSSVIIK*EPQIEPTADLTG 58 kinase, ITNQLI Ser/Thr TCK (non- receptor) 38Cytoskeletal TUBA1B; %370; 370; VGINYQPPTVVPGGDLAK*V 59 protein TUBA3D;370; 370; QR TUBA4A; 370; 370; TUBA1A; 369 TUBA1C; TUBA8; TUBA3C 39Ubiquitin UBA52; 11; %11 TLTGK*TITLEVEPSDTIENV K 60 conjugatingubiquitin system 40 Ubiquitin UBA52; 6; %6; 6 MQIFVK*TLTGK 61conjugating ubiquitin; system L0C388720 41 Ubiquitin UBA52; 29; %29;AK*IQDKEGIPPDQQR 62 conjugating ubiquitin; 29; system L0C388720; 106Gm7866 42 Ubiquitin UBA52; 29, 33; AK*IQDK*EGIPPDQQR 63 conjugatingubiquitin; %29, system L0C388720; %33; 29, Gm7866 33; 106, 110 43Ubiquitin UBA52 33; %33; IQDK*EGIPPDQQR 64 conjugating ubiquitin; 33;system L0C388720; 110 Gm7866 44 Ubiquitin UBA52; 33; %33;AKIQDK*EGIPPDQQR 65 conjugating ubiquitin; 33; system L0C388720; 110Gm7866 45 Ubiquitin UBA52; 48; %48; LIFAGK*QLEDGR 66 conjugatingubiquitin; 48; system L0C388720; 125 Gm7866 46 Ubiquitin UBA52; 48; %48;LIFAGK*QLEDGRTLSDYNI 67 conjugating ubiquitin; 48; QK system L0C388720;125 Gm7866 47 Ubiquitin UBA52; 48; %48; LIFAGK*QLEDGRTLSDYNI 68conjugating ubiquitin; 48; 125 QKESTL HLVLR system L0C388720; Gm7866 48Ubiquitin UBA52; 63; %63; TLSDYNIQK*ESTLHLVLR 69 conjugating ubiquitin;63; system LOC388720; 63; 140 OTTMUSGOO 000001634; Gm7866 49Mitochondrial 1190003J15 67 CPGLLTPSQIKPGTYK*LFFD 70 protein Rik TER 50Unassigned 1300002K09 223 SM#LEAHQAKHVK*QLLSK 71 Rik PR 51 Adaptor/14-3-3 eta; 49; 49; NLLSVAYK*NVVGAR 72 scaffold 14-3-3 49; 50 gamma; 14-3-3 zeta; 14-3-3 beta 52 Enzyme, misc. 1-Cys PRX 198KGESVM#VVPTLSEEEAK*Q 73 CFPK 53 Enzyme, misc. 1-Cys PRX 198KGESVMVVPTLSEEEAK*QC 74 FPK 54 Enzyme, misc. 1-Cys PRX 208 GVFTK*ELPSGK75 55 Receptor, ABCA3 503 TVVGK*EEEGSDPEK 76 channel, transporter orcell surface protein 56 Receptor, ABCA3 503, 512 TVVGK*EEEGSDPEK*ALR 77channel, transporter or cell surface protein 57 Receptor, ABCA3 1620SEGK*QDALEEFK 78 channel, transporter or cell surface protein 58Unassigned ABCB11 935 EILEK*AGQITNEALSNIR 79 59 Unassigned ABCB11 935MLTGFASQDKEILEK*AGQ 80 ITNEAL SNIR 60 Unassigned ABCB11 935M#LTGFASQDKEILEK*AGQ 81 ITNEAL SNIR 61 Receptor, ABCC2 491 KIQVQNM#K*NK82 channel, transporter or cell surface protein 62 Receptor, ABCC2 491IQVQNMK*NK 83 channel, transporter or cell surface protein 63 Adhesionor ABHD2 57 FLLK*SCPLLTK 84 extracellular matrix protein 64 TranslationAC078817.18- 136; 136 GKYK*EETIEK 85 1; RPL26 65 Enzyme, misc. ACAA1b;292; 292 RSK*AEELGLPILGVLR 86 ACAA1 66 Enzyme, misc. ACOX1 488IQPQQVAVWPTLVDINSLDSLTEAY 87 K*LR 67 Enzyme, misc. ACSL5 361 VYDK*VONEAK88 68 Enzyme, misc. ACSL5 616 NQCVK*EAILEDLQK 89 69 Enzyme, misc. ACSL5675 FFQTQIK*SLYESIEE 90 70 Cytoskeletal ACTG2; 193; 193; DLTDYLMK*ILTER91 protein ACTC1; 193; 191; ACTA1; 192; 195 ACTB; ACTBL2; ACTG1 71Cytoskeletal ACTG2; 328; 328; EITALAPSTM#K1K 92 protein ACTC1; 328; 326;ACTA1; 330 ACTB; ACTG1 72 Enzyme, misc. ADCY3 297 HVADEMLKDMKK* 93 73Mitochondrial ADH1C 40 IK*MVATGVCR 94 protein 74 Mitochondrial ADH1C 105ICK*HPESNFCSR 95 protein 75 Mitochondrial ADH1C 169IDGASPLDK*VCLIGCGFSTG 96 protein YGSA VK 76 Mitochondrial ADH1C 186IDGASPLDKVCLIGCGFSTG 97 protein YGSAV K*VAK 77 Mitochondrial ADH1C 316TWK*GAIFGGFK 98 protein 78 Mitochondrial ADH1C 339 LVADFMAK*K 99 protein79 Kinase (non- ADK 110 AATFFGCIGIDK*FGEILK 100 protein) 80 Kinase (non-ADK 255 EQGFETK*DIK 101 protein) 81 Kinase (non- ADK 357 TGCTFPEK*PDFH102 protein) 82 Enzyme, misc. AKR1C1 225 EK*QWVDOSSPVLLDNPVL 103 GSMAK83 Enzyme, misc. AKR1C1 312 YISGSSFK*DHPDFPFWDEY 104 84 Enzyme, misc.ALAD 87 VPK*DEQGSAADSEDSPTIE 105 AVR 85 Enzyme, misc. ALAD 87CVLIFGVPSRVPK*DEQGSA 106 ADSED SPTIEAVR 86 Enzyme, misc. ALAD 184AALLK*HGLGNR 107 87 Receptor, albumin 460 VGTK*CCTLPEDQR 108 channel,transporter or cell surface protein 88 Unassigned ALDH16A1 603RK*PVLTSQLER 109 89 Enzyme, misc. ALDH1A1 434 ANNTTYGLAAGLFTK*DLD K 11090 Enzyme, misc. ALDH1A1 434 RANNTTYGLAAGLFTK*DL 111 91 Enzyme, misc.ALDH1A2 338 IFVEESIYEEFVK* 112 92 Unassigned Aldh1a7 435ANNTTYGLAAGVFTK*DLD 113 93 Unassigned Aldh1a7 499 TVAMQISQK*NS 114 94Enzyme, misc. Aldh1a7; 91; 90 LLNK*LADLMERDR 115 ALDH1A 95 Enzyme, misc.Aldh1a7; 91; 90 LLNK*LADLMER 116 ALDH1A 96 Enzyme, misc. Aldh1a7; 255;254 LIK*EAAGK 117 ALDH1A 97 Enzyme, misc. Aldh1a7; 378; 377WGNK*GFFVQPTVFSNVTD 118 ALDH1A 98 Enzyme, misc. Aldh1a7; 378; 377WGNK*GFFVQPTVFSNVTD 119 ALDH1A 99 Enzyme, misc. Aldh1a7; 398; 397IAK*EEIFGPVQQIMK 120 ALDH1A 100 Enzyme, misc. ALDH3A2 296 LQSLLK*GQK 121101 Enzyme, misc. ALDH7A1 424 FQDEEEVFEWNNEVK*QG 122 LSSSIFT K 102Enzyme, misc. ALDOB 47 IK*VENTEENRR 123 103 Enzyme, misc. ALDOB 107GIVVGIK*LDQGGAPLAGTN 124 104 Enzyme, misc. ALDOB 107GIVVGIK*LDQGGAPLAGTN 125 KETTIQ GLDGLSER 105 Enzyme, misc. ALDOB 120LDQGGAPLAGTNK*ETTIQG 126 LDGLS ER 106 Enzyme, misc. ALDOB 329 ATQEAFMK*R127 107 Adhesion or AMFR 573 FSK*SADER 128 extracellular matrix protein108 Adhesion or AMFR 600 FLNK*SSEDDGASER 129 extracellular matrixprotein 109 Calcium- ANXA6 477 AINEAYK*EDYHK 130 binding protein 110Unassigned Apoc1 60 AAIEHIK*QK 131 111 Unassigned ApoE 105 LGK*EVQAAQAR132 112 Unassigned ApoE 252 SK*MEEQTQQIR 133 113 Unassigned APOL3 232GMK*EVLDQSGPR 134 114 Unassigned Apol9b 184 IVNK*IPQATR 135 115 Enzyme,misc. ARG1 26 GGVEK*GPAALR 136 116 Enzyme, misc. ARG1 205YFSMTEVDK*LGIGK 137 117 Unassigned ARIH1 314 QFCFNCGENWHDPVK*CK 138 118Enzyme, misc. ASL 43 HLWNVDVQGSK*AYSR 139 119 Endoplasmic ASS1 112EGAK*YVSHGATGK 140 reticulum or golgi 120 Endoplasmic ASS1 121YVSHGATGK*GNDQVR 141 reticulum or golgi 121 Endoplasmic ASS1 340HCIQK*SQERVEGK 142 reticulum or golgi 122 Endoplasmic ASS1 340HCIQK*SQER 143 reticulum or golgi 123 Chromatin, ASXL2 325KVELWK*EQFFENYYGOSS 144 DNA-

binding, DNA repair or DNA 124 Unknown AUP1 250 VQQLVAK*ELGQIGTR 145function 125 Unknown BAT3 56 EH IAASVSIPSEK*QR 146 function 126Unassigned BC066028 365, 368 THGRAK*SYK*CGECGK 147 127 TranscriptionalBCoR-like 1491; 1464 LIVNK*NAGETLLQR 148 regulator 1; BCoR 128 Enzyme,misc. BHMT; 283; 274 WDIQK*YAR 149 BHMT2 129 Ubiquitin BRAP 380LVASK*TDGK 150 conjugating system 130 Unassigned C4orf34 83GSSLPGK*PSSPHSGQDPPAP 151 PVD 131 Enzyme, misc. CA3 39 D I K*H 152DPSLQPWSASYDPGSAK 132 Enzyme, misc. CA3 57 D I KH 153DPSLQPWSASYDPGSAK*TIL NNGK 133 Endoplasmic catalase 242 TDQGIK*NLPVGEAGR154 reticulum or golgi 134 Enzyme, misc. CBS 386 FLSDK*WMLQK 155 135Chaperone CCT-alpha 126 LACK*EAVR 156 136 Chaperone CCT-alpha 541DDK*HGSYENAVHSGALDD 157 137 Chaperone CCT-theta 533 VDQIIMAKPAGGPK*PPSGK158

138 Chaperone CCT-theta 538 PAGGPKPPSGK*KDWDDDQ 159 139 ChaperoneCCT-theta 539 VDQIIMAKPAGGPKPPSGKK 160

140 Unassigned CHIC1 179 SIQK*LLEWENNR 161 141 Unknown CIRH1A 642, 645,RTTHGFK*MSK*IYK* 162 function 648 142 Cytoskeletal claudin 3 216STGPGTGTGTAYDRK*DYV 163 protein 143 Unassigned CLIC4 202 LH IVKVVAK* 164144 Vesicle CLTC 629 AH IAQLCEK*AGLLQR 165 145 Vesicle CLTC 1450AVNYFSK*VK 166 146 Vesicle CLTC 1452 VK*QLPLVKPYLR 167 147 MitochondrialCPS1 307 EPLFG ISTGN I ITG 168 protein LAAGAK*SYK 148 Mitochondrial CPS1310 SYK*MSMANR 169 protein 149 Mitochondrial CPS1 560QLFSDKLNEINEK*IAPSFAVE 170 protein SMED ALK 150 Mitochondrial CPS1 772TSACFEPSLDYMVTK*IPR 171 protein 151 Mitochondrial CPS1 1100SIFSAVLDELK*VAQAPWK 172 protein 152 Mitochondrial CPS1 1183EVEMDAVGK*EGR 173 protein 153 Mitochondrial CPS1 1269SFPFVSK*TLGVDFIDVATK 174 protein 154 Enzyme, misc. CPT1A 195YLESVRPLMK*EGDFQR 175 155 Enzyme, misc. CRAD2 64 VLAACLTEK*GAEQLR 176156 Enzyme, misc. CRAD2 224 LSHSIEK*LWDQTSSEVKEV 177 YDKNF LDSYIK 157Cell cycle CTH 47 AVVLPISLATTFK*QDFPGQS 178 regulation SGFE YSR 158 Cellcycle CTH 72 NCLEK*AVAALDGAK 179 regulation 159 Adhesion or CTNNB1 671M#SEDKPQDYK*K 180 extracellular matrix protein 160 Adhesion or CTNNB1671 MSEDKPQDYK*K 181 extracellular matrix protein 161 Adaptor/ CTNND1517 MEIVDHALHALTDEVIIPHS 182 scaffold GWERE PNEDCK*PR 162 Adaptor/CTNND1 517 M#EIVDHALHALTDEVIIPHS 183 scaffold GWER EPNEDCK*PR 163Adaptor/ CTNND1 710 SALRQEK*ALSAIAELLTSEH 184 scaffold ER 164 Receptor,Cx32 244 LSPEYK*QNEINK 185 channel, transporter or cell surface protein165 Receptor, Cx32 276 SPGTGAGLAEK*SDR 186 channel, transporter or cellsurface protein 166 Receptor, Cx32 276 RSPGTGAGLAEK*SDR 187 channel,transporter or cell surface protein 167 Adhesion or CXADR 271YEK*EVHHDIR 188 extracellular matrix protein 168 Unassigned CYB5A 38VYDLTK*FLEEHPGGEEVLR 189 169 Enzyme, CYB5R3 240 LWYTVDK*APDAWDYSQG 190misc. FVNEE M#IR 170 Enzyme, CYP1A1; 97; 94 IGSTPVVVLSGLNTIK*QALV R 191misc. CYP1A2 171 Enzyme, CYP1A2 250 YLPNPALK*R 192 misc. 172 Enzyme,CYP1A2 276 TVQEHYQDFNK*NSIQDITSA 193 misc. LFK 173 Enzyme, CYP1A2 294HSENYK*DNGGLIPEEK 194 misc. 174 Enzyme, CYP1A2 401 DTSLNGFHIPK*ER 195misc. 175 Enzyme, Cyp2a12 250 DSHKLEDFMIQK*VK 196 misc. 176 Enzyme,Cyp2a12 252 VK*QNQSTLDPNSPR 197 misc. 177 Endoplasmic CYP2A7 32LSGK*LPPGPTPLPFVGNFLQ 198 reticulum or LNTE QM#YNSLM#K golgi 178Endoplasmic CYP2A7 32 LSGK*LPPGPTPLPFVGNFLQ 199 reticulum or LNTEQMYNSLM#K golgi 179 Endoplasmic CYP2A7 32 LSGK*LPPGPTPLPFVGNFLQ 200reticulum or LNTE QMYNSLMK golgi 180 Endoplasmic CYP2A7; 239; 239HLPGPQQQAFK*ELQGLEDFI 201 reticulum or Cyp2a5 TK golgi 181 EndoplasmicCYP2A7; 342; 342 NRQPK*YEDR 202 reticulum or Cyp2a5 golgi 182Endoplasmic CYP2A7; 348; 348 MK*MPYTEAVIHEIQR 203 reticulum or Cyp2a5golgi 183 Endoplasmic CYP2A7; 409; 409 FFSNPK*DFNPK 204 reticulum orCyp2a5 golgi 184 Endoplasmic CYP2A7; 250; 250; ELQGLEDFITK*K 205reticulum or Cyp2a5; 35 golgi Cyp2a21- ps 185 Enzyme, CYP2B1; 346;TK*MPYTDAVIHEIQR 206 misc. Cyp2b9; 345; Cyp2b13 345 186 Enzyme, CYP2C19;432; KSDYFMPFSTGK*R 207 misc. Cyp2c29; 331; CYP2C9; 432; Cyp2c54; 432;Cyp2c50 432 187 Enzyme, CYP2C19; 432; SDYFMPFSTGK*R 208 misc. Cyp2c29;331; CYP2C9; 432; Cyp2c54; 432; Cyp2c50 432 188 Enzyme, CYP2C19; 84; 84KPTVVLHGYEAVK*EALVD 209 misc. Cyp2c50 HGEEFA GR 189 Unassigned Cyp2c29298 GTTVITSLSSVLHDSK*EFPN 210 PEM# FDPGHFLNGNGNFK 190 Enzyme, Cyp2c39399 GTTVVTSLTSVLHDSK*EFP 211 misc. NPELF DPGHFLDANGNFK 191 Enzyme,Cyp2c39; 270; DFIDYYLIK*QK 212 misc. Cyp2c29; 169; CYP2C9 270 192Enzyme, Cyp2c40 375 YIDLGPNGVVHEVTCDTK*FR 213 misc. 193 Enzyme, Cyp2c40;110; 110 GK*GIGFSHGNVWK 214 misc. L0C1000483 23 194 Enzyme, Cyp2c40;154; 154 VQEEAQWLM#K*ELKK 215 misc. L0C1000483 23 195 Enzyme, Cyp2c40;154; 154 VQEEAQWLMK*ELKK 216 misc. L0C1000483 23 196 Enzyme, Cyp2c40;154; 154 VQEEAQWLMK*ELK 217 misc. L0C1000483 23 197 Enzyme, Cyp2c40;154; 154 VQEEAQWLM#K*ELK 218 misc. L0C1000483 23 198 Enzyme, Cyp2c40;157; 157 VQEEAQWLM#KELK* 219 misc. L0C1000483 23 199 Enzyme, Cyp2c40;157; 157 VQEEAQWLMKELK*K 220 misc. L0C1000483 23 200 Enzyme, Cyp2c40;249; 249 IK*EHEESLDVTNPR 221 misc. LOCI000483 23 201 Enzyme, Cyp2c54 84KPTVVLHGYEAVK*EALVD 222 misc. HGDVF AGR 202 Unassigned Cyp2c70 234FLK*DVTQQK 223 203 Unassigned Cyp2c70 234 FLK*DVTQQKK 224 204 UnassignedCyp2c70 252 HQK*SLDLSNPQDFIDYFLIK 225 205 Unassigned Cyp2d10 414GSILIPNM#SSVLKDETVWEK 226 *PLR 206 Enzyme, CYP2D2 414GTTLIPNLSSVLKDETVWEK* 227 misc. PLR 207 Unassigned Cyp2d40 252GTTLICNLSSVLKDETVWEK 228 *PLR 208 Endoplasmic CYP2E1 59 SLTK*LAK 229reticulum or golgi 209 Endoplasmic CYP2E1 84 IVVLHGYK*AVK 230 reticulumor golgi 210 Endoplasmic CYP2E1 84 RIVVLHGYK*AVK 231 reticulum or golgi211 Endoplasmic CYP2E1 87 AVK*EVLLNHKNEFSGR 232 reticulum or golgi 212Endoplasmic CYP2E1 94 EVLLNHK*NEFSGR 233 reticulum or golgi 213Endoplasmic CYP2E1 110 GDIPVFQEYK*NK 234 reticulum or golgi 214Endoplasmic CYP2E1 112 NK*GlIFNNGPTWK 235 reticulum or golgi 215Endoplasmic CYP2E1 123 GlIFNNGPTWK*DVR 236 reticulum or golgi 216Endoplasmic CYP2E1 140 DWGM#GK*QGNEAR 237 reticulum or golgi 217Endoplasmic CYP2E1 140 DWGMGK*QGNEAR 238 reticulum or golgi 218Endoplasmic CYP2E1 159 EAHFLVEELK*K 239 reticulum or golgi 219Endoplasmic CYP2E1 162 TK*GQPFDPTFLIGCAPCNVI 240 reticulum or ADILF NKgolgi 220 Endoplasmic CYP2E1 255 AKEHLK*SLDINCPR 241 reticulum or golgi221 Endoplasmic CYP2E1 255 EHLK*SLDINCPR 242 reticulum or golgi 222Endoplasmic CYP2E1 275 DVTDCLLIEMEK*EK 243 reticulum or golgi 223Endoplasmic CYP2E1 428 YSDYFK*AFSAGK 244 reticulum or golgi 224Endoplasmic CYP2E1 428 YSDYFK*AFSAGKR 245 reticulum or golgi 225Endoplasmic CYP2E1 467 SLVDPK*DIDLSPVTIGFGSIP R 246 reticulum or golgi226 Receptor, CYP3A4 35 K*QGIPGPTPLPFLGTVLNYY K 247 channel, transporteror cell surface protein 227 Receptor, CYP3A4 380 FCKK*DVELNGVYIPK 248channel, transporter or cell surface protein 228 Receptor, CYP3A4 425ENK*GSIDPYLYMPFGIGPR 249 channel, transporter or cell surface protein229 Receptor, CYP3A4 425 ENK*GSIDPYLYM#PFGIGPR 250 channel, transporteror cell surface protein 230 Receptor, CYP3A4 425 FSKENK*GSIDPYLYMPFGIG251 channel, PR transporter or cell surface protein 231 Receptor, CYP3A4477 VMQNFSFQPCQETQIPLK*LS R 252 channel, transporter or cell surfaceprotein 232 Receptor, CYP3A43; 421; 422; FSK*ENK 253 channel, CYP3A5;422; 558; transporter CYP3A7; 657; 422; or cell UVRAG; 422 surfaceKIAA1802; protein Cyp3a44; CYP3A4 233 Unassigned Cyp3a44 422FSK*ENKGSIDPYVYLPFGIG 254 PR 234 Unassigned Cyp3a44 488 QGILOPEK*PIVLK255 235 Unassigned Cyp3a44 493 QGILOPEKPIVLK*VVPR 256 236 Receptor,Cyp3a44; 116; 116; EFGPVGIMSK*AISISKDEEW 257 channel, CYP3A4; 16 KRtransporter L00673748 or cell surface protein 237 Receptor, CYP3A5; 96;96 NVLVK*ECFSVFTNRR 258 channel, CYP3A4 transporter or cell surfaceprotein 238 Receptor, CYP3A5; 141; 141 ALLSPTFTSGK*LK 259 channel,CYP3A4 transporter or cell surface protein 239 Receptor, CYP3A5; 488;488 QGLLQPEK*PIVLK 260 channel, CYP3A4 transporter or cell surfaceprotein 240 Enzyme, CYP3A5; 35; 35 K*QGIPGPKPLPFLGTVLNYY K 261 misc.CYP3A7 241 Enzyme, CYP3A5; 42; 42 QGIPGPK*PLPFLGTVLNYYK 262 misc. CYP3A7242 Receptor, CYP3A5; 96; 96; 96 NVLVK*ECFSVFTNR 263 channel, CYP3A7;transporter CYP3A4 or cell surface protein 243 Enzyme, CYP3A5; 158.LKEM#FPVIEQYGDILVK*YLR 264 misc. CYP3A7; 158: Cyp3a44 158 244 Enzyme,CYP3A5; 158; EM#FPVIEQYGDILVK*YLR 265 misc. CYP3A7; 158: Cyp3a44 158 245Enzyme, CYP3A5; 158; LKEMFPVIEQYGDILVK*YLR 266 misc. CYP3A7; 158;Cyp3a44 158 246 Receptor, CYP3A5; 143; 143; LK*EM#FPVIEQYGDILVK 267channel, CYP3A7; 143; 143 transporter Cyp3a44; or cell CYP3A4 surfaceprotein 247 Receptor, CYP3A5; 143; 143; LK*EMFPVIEQYGDILVK 268 channel,CYP3A7; 143; 143 transporter Cyp3a44; or cell CYP3A4 surface protein 248Receptor, CYP3A5; 250; 250; DSIEFFK*K 269 channel, CYP3A7; 250; 250transporter Cyp3a44; or cell CYP3A4 surface protein 249 Receptor,CYP3A7; 59; 59 GLWK*FDMECYEK 270 channel, CYP3A4 transporter or cellsurface protein 250 Endoplasmic CYP4A11 252 LAK*QACQLAHDHTDGVIK 271reticulum or golgi 251 Enzyme, CYP51A1 436 YLQDNPASGEK*FAYVPFGAGR 272misc. 252 Enzyme, CYP51A1 436 LDFNPDRYLQDNPASGEK*F 273 misc. AYVP FGAGR253 Endoplasmic Cyp7a1 127 SIDPSDGNTTENINK*TFNK 274 reticulum or golgi254 Enzyme, misc. CYP8B1 366 VVQEDYVLK*MASGQEYQ IR 275 255 Lipid DBI 50QATVGDVNTDRPGLLDLK*GK 276 binding protein 256 Adhesion or desmoplakin152 QMGQPCDAYQK*R 277 extracellular matrix protein 257 Adhesion ordesmoplakin 166 ALYK*AISVPR 278 extracellular matrix protein 258Adhesion or desmoplakin 249 WQLDK*IK 279 extracellular matrix protein259 Enzyme, misc. Diminuto 446 VK*HFEAR 280 260 Unassigned DNAJA2 158SGAVQK*CSACR 281 261 Enzyme, misc. DPYD 875 VAELMGQK*LPSFGPYLEQR 282 262Adhesion or DSC2 838 LGDK*VQFCHTDDNQK 283 extracellular matrix protein263 Receptor, DYSF 1612 ISIGK*K 284 channel, transporter or cell surfaceprotein 264 Translation eEF-2 271 YFDPANGK*FSK 285 265 Translation eEF-2274 FSK*SANSPDGK 286 266 Translation elF3C 860 TEPTAQQNLALQLAEK*LGS 287LVENN ER 267 Translation elF3-theta 420 EQPEK*EPELQQYVPQLQNN 288 268Translation elF3-theta 775 QSVYEEK*LKQFEER 289 269 Vesicle epsin 1 107ENMYAVQTLK*DFQYVDR 290 protein DGKDQ GVNVR 270 Enzyme, misc. esterase D17 CFGGLQK*VFEHSSVELK 291 271 Lipid FABP1 20 YQLQSQENFEPFMK*AIGLP 292binding EDLIQ K protein 272 Lipid FABP1 31 AIGLPEDLIQK*GK 293 bindingprotein 273 Lipid FABP1 36 GKDIK*GVSEIVHEGK 294 binding protein 274Lipid FABP1 36 DIK*GVSEIVHEGK 295 binding protein 275 Lipid FABP1 46GVSEIVHEGK*K 296 binding protein 276 Lipid FABP1 80 VK*AVVKLEGDNK 297binding protein 277 Lipid FABP1 84 AVVK*LEGDNK 298 binding protein 278Lipid FABP1 99 M#VTTFKGIK* 299 binding protein 279 Receptor, FADS2 28WEEIQK*HNLR 300 channel, transporter or cell surface protein 280Receptor, FADS2 87 FLK*PLLIGELAPEEPSLDR 301 channel, transporter or cellsurface protein 281 Enzyme FAH 186 RPMGQMRPDNSK*PPVYGA 302 misc. CR 282Enzyme, FBXL11 808 AKIRGSYLTVTLQRPTK* 303 misc. 283 Enzyme, FDPS 293QILEENYGQK*DPEKVAR 304 misc. 284 Enzyme, FDPS 297 QILEENYGQKDPEK*VAR 305misc. 285 Enzyme, FM03 209 VLVIGLGNSGCDIAAELSHV 306 misc. AQK*V TISSR286 Enzyme, Fmo5 259 NNYMEK*QMNQR 307 misc. 287 Unassigned FUND2 123SK*AEEVVSFVKKNVLVTGG 308 FFGG FLLGMAS 288 Apoptosis G6PI 226TFTTQETITNAETAK*EWFLE 309 AAKD PSAVAK 289 Mitochondria! GAPDH; 212; 213;GAAQNIIPASTGAAK*AVGK 310 protein Gm10291; 195; 219; Gm13882; 231EG622339; L00638833 290 Mitochondria! GAPDH; 256; 331; LEKPAKYDDIK*K 311protein LOC676923; 239; 259; Gm13882; 275; 476 LOCI00043839; L00638833;L00675602 291 Enzyme, GDA 133 TLK*NGTTTACYFGTIHTDSS 312 misc. LILAEITDKFGQR 292 G protein or G- 33 VSK*ASADLMSYCEEHAR 313 regulatorgamma(12) 293 Enzyme, GLUL 95 KDPNK*LVLCEVFK 314 misc. 294 Enzyme, GNMT96 YALK*ER 315 misc. 295 Enzyme, GSTA2; 141; 141; VLK*SHGQDYLVGNR 316misc. GSTA5; 141 GSTA3 296 Enzyme, GSTA3 64 SDGSLM#FQQVPMVEIDGM# 317misc. K*LV QTK 297 Enzyme, GSTA3 64 SDGSLMFQQVPM#VEIDGM# 318 misc. K*LVQTK 298 Enzyme, GSTA3 64 SDGSLMFQQVPM#VEIDGMK*LVQ 319 misc. TK 299Enzyme, GSTM1 198 ISAYMK*SSR 320 misc. 300 Enzyme, GSTM1; 51; 51; 52FK*LGLDFPNLPYLIDGSHK 321 misc. GSTM5; GSTM4 301 Enzyme, GSTM1; 68; 68;69 LGLDFPNLPYLIDGSHK*IT 322 misc. GSTM5; QSNAIL R GSTM4 302 Enzyme,GSTP1 127 ALPGHLK*PFETLLSQNQGG K 323 misc. 303 Unassigned Gstt3 218, 229AK*DM#PPLMDPALK* 324 304 Enzyme, Gulo 332 AMLEAHPK*VVAHYPVEVR 325 misc.305 Enzyme, Gulo 332 AM#LEAHPK*VVAHYPVEV R 326 misc. 306 Chromatin, H1F059 SHYK*VGENADSQIK 327 DNA- binding, DNA repair or DNA replicationprotein 307 Unassigned H2AE 126 VTIAQGGVLPNIQAVLLPKK 328 TESHH K*PK 308Chromatin, H2AX 127 KSSATVGPK*APAVGK 329 DNA-binding, DNA repair or DNAreplication protein 309 Chromatin, H2B; 5; 6; 6 PEPAK*SAPAPK 330 DNA-H2B2E; binding, H2B1C DNA repair or DNA replication protein 310Receptor, HBA1 12 SNIK*AAWGK 331 channel, transporter or cell surfaceprotein 311 Receptor, HBA1 17 AAWGK*IGGHGAEYGAEAL 332 channel, ERtransporter or cell surface protein 312 Receptor, HBA1 41M#FASFPTTK*TYFPHFDVSH 333 channel, GSAQ VK transporter or cell surfaceprotein 313 Receptor, HBA1 41 MFASFPTTK*TYFPHFDVSH 334 channel, GSAQ VKtransporter or cell surface protein 314 Receptor, HBA1 57TYFPHFDVSHGSAQVK*GHG 335 channel, transporter or cell 315 Receptor, HBA191 VADALASAAGHLDDLPGA 336 channel, LSALSD LHAHK*LR transporter or cellsurface protein 316 Receptor, HBA1 91 KVADALASAAGHLDDLPG 337 channel,ALSALS DLHAHK*LR transporter or cell surface protein 317 Receptor, HBB17 SAVSCLWAK*VNPDEVGGE 338 channel, ALGR transporter or cell surfaceprotein 318 Receptor, HBB 59 YFDSFGDLSSASAIMGNPK* 339 channel,transporter or cell 319 Receptor, HBB 82 NLDNLK*GTFASLSELHCDK 340channel, LHVDP ENFR transporter or cell surface 320 Receptor, HBD 17AAVSCLWGK*VNSDEVGGE 341 channel, transporter or cell 321 Receptor, HBD59 YFDSFGDLSSASAIM#GNAK* 342 channel, transporter or cell 322 Receptor,HBD 82 VITAFNDGLNHLDSLK*GTF 343 channel, ASLSEL HCDKLHVDPENFRtransporter or cell 323 Enzyme, HGD 71 ILPSVSHK*PFESIDQGHVTH 344 misc.NWDE VGPDPNQLR 324 Enzyme, HGD 252 FQGK*LFACK 345 misc. 325 RNA hnRNPA/B 88 MFVGGLSWDTSK*K 346 processing 326 RNA hnRNP A/B 89 MFVGGLSWDTSKK*347 processing 327 RNA hnRNP A/B 237 VAQPK*EVYQQQQYGSGGR 348 processing328 Enzyme, HPD 62 EVVSHVIK*QGK 349 misc. 329 Enzyme, HPD 126IVREPWVEQDK*FGK 350 misc. 330 Enzyme, HPD 129 IVREPWVEQDKFGK*VK 351misc. 331 Enzyme, HPD 131 VK*FAVLQTYGDTTHTLVEK 352 misc. 332 Enzyme, HPD131 IVREPWVEQDKFGKVK* 353 misc. 333 Enzyme, HPD 236SIVVTNYEESIK*MPINEPAPG R 354 misc. 334 Enzyme, HPD 247K*KSQIQEYVDYNGGAGVQ 355 misc. HIALK 335 Enzyme, HPD 248KK*SQIQEYVDYNGGAGVQ 356 misc. HIALK 336 Enzyme, HPD 269SQIQEYVDYNGGAGVQHIAL 357 misc. K*TED IITAIR 337 Enzyme, HPD 296ERGTEFLAAPSSYYK*LLR 358 misc. 338 Enzyme, HPD 368 HNHQGFGAGNFNSLFK*AF359 misc. EEEQA LR 339 Enzyme, HRSP12 66 NLGEILK*AAGCDFNNVVK 360 misc.340 Chaperone HSC70 108 VQVEYK*GETK 361 341 Chaperone HSC70 512LSK*EDIER 362 342 Chaperone HSC70 524 MVQEAEK*YKAEDEK 363 343 ChaperoneHSC70 524 MVQEAEK*YKAEDEKQR 364 344 Chaperone HSC70 524M#VQEAEK*YKAEDEKQR 365 345 Chaperone HSC70 583 ILDKCNEIISWLDK*NQTAE 366KEEFEH QQK 346 Chaperone HSC70 583 ILDKCNEIISWLDK*NQTAE 367 KEEFEHQQKELEK 347 Chaperone HSC70

345; 348 IPK*IQK 368 348 Endoplasmic HSD11B1 73 SEEGLQK*VVSR 369reticulum or golgi 349 Receptor, IFITM3 24 IK*EEYEVAEMGAPHGSASVR 370channel, transporter or cell surface protein 350 Receptor, IFITM3 24IK*EEYEVAEM#GAPHGSASVR 371 channel, transporter or cell surface protein351 Kinase IPPK 42, 43, 64 K*K*TSEEILQHLQNIVDFGKNVMK* 372 (non- protein)352 G protein IQGAP2 1024 AWVNQLETQTGEASK*LPY 373 or DVTTE QALTYPEVKregulator 353 G protein IQGAP2 1354 TPEEGK*QSQAVIEDAR 374 or regulator354 RNA IREB1 79 NIEVPFK*PAR 375 processing 355 Ubiquitin ITCH 192VSTNGSEDPEVAASGENK*R 376 conjugating system 356 Ubiquitin ITCH 407FIYGNQDLFATSQNKEFDPL 377 conjugating GPLPP GWEK*R system 357 UnassignedITM2B 13 VTFNSALAQK*EAK 378 358 Unassigned JOSD1 180GK*NCELLLVVPEEVEAHQS 379 WR 359 Enzyme, KHK 159 IEEHNAK*QPLPQK 380 misc.360 Unknown KIAA1033 1089 AVAK*QQNVOSTSQDEK 381 function 361Cytoskeletal lamin A/C 270 TYSAK*LDNAR 382 protein 362 CytoskeletalLamin B1 124, 134 K*ESDLSGAQIK*LR 383 protein 363 Vesicle LAPTM4A 224IPEK*EPPPPYLPA 384 protein 364 Calcium- LETM1 715 VIDLVNKEDVQISTTQVAEI385 binding VATLEK *EEK protein 365 Receptor, LISCH 538 LLEEALK*K 386channel, transporter or cell surface protein 366 Unassigned LOC100044463, 73; MQANNAK*AVSARTEAIK*A 387 94; Gm12508 176, 186 LVK 367 UnassignedLOCI000483 247 SYLLEK*IKEHEESLDVTNPR 388 23 368 Unassigned LOCI000483343 K*HMPYTNAMVHEVQR 389 23 369 Unassigned LOC1000483 375YVDLGPTSLVHEVTCDTK*F R 390 23 370 Unknown LOC144100 742 DQPQHLEK*ITCQQR391 function 371 G protein or LOC435565; 406; 400; TLLK*EICLRN 392regulator EG240327; 407 ligp1 372 Cytoskeletal MARCKS 10TAAK*GEATAERPGEAAVAS 393 protein SPSK 373 Enzyme, MAT1A 54QDPNAK*VACETVCK 394 misc. 374 Enzyme, MAT1A 89 DTIK*HIGYDDSAK 395 misc.375 Enzyme, MAT1A 98 HIGYDDSAK*GFDFK 396 misc. 376 Enzyme, MAT1A 352ELLEVVNK*NFDLRPGVIVR 397 misc. 377 Enzyme, MAT1A 368 DLDLK*KPIYQK 398misc. 378 Enzyme, MAT1A 374 KPIYQK*TACYGHFGR 399 misc. 379 Enzyme, MAT1A374 DLDLKKPIYQK*TACYGHFG R 400 misc. 380 Unassigned MBD2 193SDVYYFSPSGKKFRSK* 401 381 Unassigned MCT1 467 EGKEDEASTDVDEK*PKETM 402382 Unassigned MCT1 469 EGKEDEASTDVDEKPK*ETM 403 383 Enzyme, Mettl7b 241WLPVGPHIM#GK*AVK 404 misc. 384 Enzyme, Mettl7b 241 WLPVGPHIMGK*AVK 405misc. 385 Enzyme, MGST1 59 VFANPEDCAGFGKGENAK* K 406 misc. 386 Enzyme,MGST1 60 VFANPEDCAGFGKGENAKK* 407 misc. 387 Transcriptional MORF4L1 117ELQK*ANQEQYAEGK 408 regulator 388 Mitochondrial MOSC1 313LCDPSEQALYGK*LPIFGQY 409 protein FALEN PGTIR 389 Adaptor/ MPP5 553DYHFVSRQAFEADIAAGKFI 410 scaffold EHGEF EK*NLYGTSIDSVR 390 Receptor,MT2A 20 MDPNCSCASDGSCSCAGAC 411 channel, K*CK transporter or cellsurface protein 391 Mitochondrial MTX1 41 IHK*TSNPWQSPSGTLPALR 412protein 392 Ubiquitin NEDD8 54 QMNDEK*TAADYK 413 conjugating system 393Enzyme, NGLY1 130 KVQFSQHPAAAK*LPLEQSE 414 misc. DPAG LIR 394 Enzyme,NGLY1 130 VQFSQHPAAAK*LPLEQSE 415 misc. DPAGLI R 395 Enzyme, NKEF-A 109QGGLGPMNIPLISDPK*R 416 misc. 396 Kinase NME2 56 QHYIDLK*DRPFFPGLVK 417(non- protein) 397 Adaptor/ NOSTRIN 417 AESK*APAGGQNNPSSSPSG 418scaffold STVS QASK 398 Enzyme, NQ02 23 SFNGSLK*K 419 misc. 399 VesicleNSFL1C 127 GAK*EHGAVAVER 420 protein 400 Receptor, NUP214 686STQTAPSSAPSTGQK*SPRV 421 channel, NPPV transporter PKSGSSQAKALQPPVTEK orcell surface protein 401 Enzyme, p67phox 354 EPKELKLSVPM#PYM#LK* 422misc. 402 RNA PABP 1 284 KFEQMK*QDR 423 processing 403 Enzyme, PAH 49EEVGALAK*VLR 424 misc. 404 Enzyme, PAH 95 SKPVLGSIIK*SLR 425 misc. 405Enzyme, PAH 149 TIQELDRFANQILSYGAELD 426 misc. ADHPG FK*DPVYR 406Enzyme, PAPSS2 174 AGEIK*GFTGIDSDYEKPET 427 misc. PECVL K 407 KinasePCK1 124 WMSEEDFEK*AFNAR 428 (non- protein) 408 Kinase PCK1 124WM#SEEDFEK*AFNAR 429 (non- protein) 409 Kinase PCK1 471 SEATAAAEHK*GK430 (non- protein) 410 Cell cycle PCM-1 1089 QQNQHPEK*PR 431 regulation411 Adaptor/ PDZK1 118 EAALNDKK*PGPGMNGAVE 432 scaffold PCAQP R 412Phosphatase PGAM1 105 AETAAK*HGEAQVK 433 413 Vesicle PICALM 324EK*QAALEEEQAR 434 protein 414 Kinase PIP5KG 97 GAIQLGIGYTVGNLSSK*PER 435(non- protein) 415 Protein PKG2 428 RSMSSWKLSK* 436 kinase, Ser/Thr(non- receptor) 416 Adhesion or plakophilin 2 134 AAAQYSSQK*SVEER 437extracellular matrix protein 417 Receptor, PMP70 260 MTIMEQK*YEGEYR 438channel, transporter or cell 418 Receptor, PMP70 576 EGGWDSVQDWMDVLSGGE439 channel, transporter or cell 419 Enzyme, PP ID; 285; 263LQPIALSCVLNIGACKLK* 440 misc. LOCI000452 51 420 Cytoskeletal profilin 169 SSFFVNGLTLGGQK*CSVIR 441 protein 421 Protease PSMA2 69SVHKVEPITK*HIGLVYSGM 442 #GPDY R 422 Protease PSMA6 102 ARYEAANWK*YK 443423 Protease PSMB5 91 ATAGAYIASQTVK*K 444 424 Protease PSMC2 116YIINVK*QFAK 445 425 Protease PSMC2 116 IINADSEDPKYIINVK*QFAK 446 426Transcriptional PSMC3 279 DAFALAK*EK 447 regulator 427 TranscriptionalPSMC3 279 DAFALAK*EKAPSIIFIDELDAI 448 regulator GTK 428 Protease PSMC648 SENDLK*ALQSVGQIVGEVL 449 429 Protease PSMC6 197 AVASQLDCNFLK*VVSSSIVD450 430 Protease PSMC6 197 AVASQLDCNFLK*VVSSSIVD 451 KYIGE SAR 431Protease PSMD13 115 SSDEAVILCK*TAIGALK 452 432 Protease PSMD4 122IIAFVGSPVEDNEK*DLVK 453 433 Transcriptional PTRF 163 NFKVM#IYQDEVK* 454regulator 434 Enzyme, PYGL 169 YEYGIFNQK*IR 455 435 Enzyme, PYGL 803AWNTM#VLK*NIAASGK 456 436 Enzyme, PYGL 803 AWNTMVLK*NIAASGK 457 437 Gprotein or Rab2 165 TASNVEEAFINTAK*EIYEK 458 regulator 438 Cytoskeletalradixin 79 KENPLQFK*FR 459 protein 439 Cytoskeletal radixin 211IAQDLEMYGVNYFEIKNK*K 460 protein 440 G protein or RALBP1 186KKPIQEPEVPQM#DAPSVK* 461 regulator 441 Unassigned RGN 233 LDPETGK*R 462442 Unassigned Rhbdd3 268 LGPGQLTWK*NSER 463 443 G protein or RhoA 135MK*QEPVKPEEGR 464 regulator 444 Unknown RNF185 105 EK*TPPRPQGQRPEPENR465 function 445 Ubiquitin RNF20 610 DSVKDKEK*GKHDDGR 466 conjugatingsystem 446 Ubiquitin RNF5 93 LK*TPPRPQGQRPAPESR 467 conjugating system447 Unassigned Rnft1 382 EKTCPLCRTVISECINK* 468 448 Translation RPL12;61; 30 ITVK*LTIQNR 469 EG633570 449 Translation RPL17 95KSAEFLLHMLK*NAESNAELK 470 450 Unassigned RPL18 78 ENK*TAVVVGTVTDDVR 471451 Translation RPL19 186 KEEIIK*TLSKEEETKK 472 452 Translation RPL19190 TLSK*EEETKK 473 453 Translation RPL19 195 TLSKEEETK*K 474 454Unassigned RPL29; 134; 134; APAK*AQASAPAQAPK 475 LOCI000444 247; 148 94;Gm1250 455 Unassigned RPL29; 151; 151; AQASAPAQAPKGAQAPK* 476 LOCI000444264; 165 94; Gm1250 456 Translation RPL3 293 IGQGYLIK*DGK 477 457Translation RPL3 299 LIK*NNASTDYDLSDK 478 458 Translation RPL4 294ILK*SPEIQR 479 459 Translation RPL4 333 LNPYAK*TMR 480 460 TranslationRPL4 364 KLEAAATALATK*SEK 481 461 Unassigned RPL9; 21; 21TILSNQTVDIPENVEITLK*GR 482

462 Unassigned RPLP2 24 YVASYLLAALGGNSSPSAKD 483 463 Enzyme, RPN1 539LK*TEGSDLCDRVSEMQK 484 misc. 464 Translation RPS10 138 SAVPPGADK*K 485465 Translation RPS10 138 RSAVPPGADK*K 486 466 Translation RPS10 138SAVPPGADK*KAEAGAGSA 487

467 Translation RPS10 138, 139 SAVPPGADK*K*AEAGAGS 488

468 Translation RPS10 139 RSAVPPGADKK* 489 469 Translation RPS10 139SAVPPGADKK*AEAGAGSA 490

470 Translation RPS12 129 DVIEEYFK*CKK 491 471 Translation RPS17 18VIIEK*YYTR 492 472 Translation RPS2; 176; 158; IGK*PHTVPCK 493 Gm8841;67; 171 EG625055; Gm5978 473 Translation RPS2; 58; 58; AEDK*EWIPVTK 494Gm8841; 54 Gm5978 474 Unassigned RPS20 34 SLEK*VCADLIR 495 475Translation RPS21 51 FNGQFK*TYGICGAIR 496 476 Translation RPS25 114NTK*GGDAPAAGEDA 497 477 Translation RPS3 214 KPLPDHVSIVEPK*DEILPT 498TPISEQ K 478 Translation RPS3 214 IGPKKPLPDHVSIVEPK*DEI 499 LPTTPI SEQK479 Translation RPS3 230 GGK*PEPPAMPQPVPTA 500 480 Translation RPS3a 45NIGK*TLVTR 501 481 Translation RPS7 10 IVK*PNGEKPDEFESGISQA 502 LLELEM#NSDLK 482 Translation RPS7 15 IVKPNGEK*PDEFESGISQA 503 LLELE M#NSDLK483 Translation RPS7 15 IVKPNGEK*PDEFESGISQA 504 LLELE MNSDLK 484Translation RRBP1 145 K*VAKVEPAVSSIVNSIQVLA 505 SK 485 Translation RRBP1166 VEPAVSSIVNSIQVLASK*SA 506 ILEATP K 486 Translation RRBP1 219, 249,KGEGAQNQGK*KGEGAQNQ 507 289 AK 487 Translation RRBP1 229, 299,KGEGAQNQAK*KGEGAQNQ 508 359, 500 AK 488 Translation RRBP1 259, 339,KGEGAQNQAK*KGEGGQNQ 509 480 489 Translation RRBP1 269KGEGGQNQAK*KGEGAQNQ 510 490 Translation RRBP1 279, 601,KGEGAQNQGK*KGEGAQNQ 511 611, 621, 631, 641, 651, 681, 491 TranslationRRBP1 369, 510 KGEGAQNQAK*KGEGVQNQ 512 492 Translation RRBP1 440, 581IEGAQNQGK*KPEGTSNQGK 513 493 Translation RRBP1 440, 581KIEGAQNQGK*KPEGTSNQG 514 494 Translation RRBP1 671 KGEGPQNQAK*KGEGAQNQ515 495 Translation RRBP1 752 TDTVANQGTK*QEGVSNQV 516 496 TranslationRRBP1 752 KTDTVANQGTK*QEGVSNQ 517 497 Translation RRBP1 823ASM#VQSQEAPK*QDAPAK 518 498 Translation RRBP1 823 ASMVQSQEAPK*QDAPAK 519499 Enzyme, SAHH 46 EMYSASKPLK*GAR 520 misc. 500 Enzyme, SAHH 166GISEETTTGVHNLYK*M#MS 521 misc. NGILK 501 Enzyme, SAHH 166GISEETTTGVHNLYK*MMSN 522 misc. GILK 502 Enzyme, SAHH 166GISEETTTGVHNLYK*MM#S 523 misc. NGILK 503 Enzyme, SAHH 166GISEETTTGVHNLYK*M#MS 524 misc. NGILK VPAINVNDSVTK 504 Enzyme, SAHH 166GISEETTTGVHNLYK*MMS 525 misc. NGILKV PAINVNDSVTK 505 Enzyme, SAHH 166GISEETTTGVHNLYK*M#M 526 misc. #SNGIL KVPAINVNDSVTK 506 Enzyme, SAHH 174GISEETTTGVHNLYKM#MSN 527 misc. GILK* 507 Enzyme, SAHH 174GISEETTTGVHNLYKMM#S 528 misc. NGILK* VPAINVNDSVTK 508 Enzyme, SAHH 186VPAINVNDSVTK*SK 529 misc. 509 Enzyme, SAHH 188 SK*FDNLYGCR 530 misc. 510Adaptor/ SAKS1 105 MLELVAQK*QR 531 scaffold 511 Lipid SEC14L2 11VGDLSPK*QEEALAK 532 binding protein 512 Lipid SEC14L2 275DQVK*QQYEHTVQVSR 533 binding protein 513 Vesicle SEC31L1 791AQGK*PVSGQESSQSPYER 534 protein 514 Unassigned SELENBP1; 342; 342QYDISNPQK*PR 535 SELENBP2 515 Protein SgK307 1148, 1153NTSLTDIQDLSSITYDQDGYF 536 kinase, K*ETS YK*TPKLK Ser/Thr (non- receptor)516 Chaperone SGTA 161 AIGIDPGYSK*AYGR 537 517 Unassigned SLC22A1 319KVPPADLK*MMCLEEDASER 538 518 Receptor, SLC26A1 32 ROPPVSQGLLETLK*AR 539channel, transporter or cell surface protein 519 Endoplasmic SLC27A5 163LK*DAVIONTR 540 reticulum or golgi 520 Endoplasmic SLC27A5 599VGMAAVK*LAPGK 541 reticulum or golgi 521 Endoplasmic SLC27A5 667EGFDVGIIADPLYILDNK*AQ 542 reticulum or TFR golgi 522 Unassigned S1c38a345 TEDTQHCGEGK*GFLQK 543 523 Unassigned S1c38a3 50 GFLQK*SPSKEPHFTDFEGK544 524 Unassigned S1c38a3 54 SPSK*EPHFTDFEGK 545 525 Unassigned Slc40a1240 AALK*VEESELK 546 526 Receptor, SLCO1A1 647 LTEK*ESECTDVCR 547channel, transporter or cell surface protein 527 Receptor, SLCO1B3 683KFTDEGNPEPVNNNGYSCV 548 channel, PSDE K*NSETPL transporter or cellsurface protein 528 Unassigned SLCO2A1 61 SSLTTIEK* 549 529 UnassignedSLCO2B1 676 TTVK*SSELQQL 550 530 Apoptosis SOD1 136 QDDLGKGGNEESTK*TGNA551 GSR 531 Endoplasmic SRP68 38 SAGGDENK*ENERPSAGSK 552 reticulum orgolgi 532 Adaptor/ ST13 355 YQSNPK*VMNLISK 553 scaffold 533 Receptor,STEAP4 97 EHYDSLTELVDYLK*GK 554 channel, transporter or cell surfaceprotein 534 Enzyme, SULT1A1 93 IPFLEFSCPGVPPGLETLK*ET 555 misc. PAPR 535Enzyme, SULT2A1 90 SPWIETDIGYSALINK*EGPR 556 misc. 536 Unassigned SYNC137 M#ASPEPLRGGDGARASRE 557 PHTE ASFPLQESESPKEAK* 537 Adaptor/ SYNE2 5243QSSLTM#DGGDVPLLEDMA 558 scaffold SGIVE LFQK*K 538 Enzyme, TALD01 258ALAGCDFLTISPK*LLGELLK 559 misc. 539 Enzyme, TALD01 265 LLGELLK*DNSK 560misc. 540 Enzyme TALD01 277 LAPALSVK*AAQTSDSEKIHL 561 misc. DEK 541Protein Titin 855 ELSATSSTQK*ITK 562 kinase, Ser/Thr (non- receptor) 542Enzyme, TKT 260 GITGIEDKEAWHGK*PLPK 563 543 Enzyme, TKT 281NMAEQIIQEIYSQVQSK*K 564 544 Unassigned TMEM59 315 SQTEEHEEAGPLPTK*VNLAH565 545 Vesicle TOLLIP 143 lAWTHITIPESLK*QGQVEDE 566 protein WYSL SGR546 Unassigned TRPM8 283, 298 NQLEK*YISERTSQDSNYGG 567 K*IPIVCFAQGGGRETLK 547 Cell cycle TSGA2 35 NEVGERHGHGK*AR 568 regulation 548Cytoskeletal TUBB2C; 216; 216; TLK*LTTPTYGDLNHLVSAT 569 protein TUBB;216; 216; MSGVT TCLR TUBB2A; 216 TUBB2B; TUBB4 549 Enzyme, TXNL1 180LYSMK*FQGPDNGQGPK 570 misc. 550 Ubiquitin UBE1 604 KPLLESGTLGTK*GNVQVVI571 conjugating PFLTE SYSSSQDPPEK system 551 Ubiquitin UBE1 627GNVQVVIPFLTESYSSSQDPP 572 conjugating EK*S IPICTLK system 552 UbiquitinUBE1 635 SIPICTLK*NFPNAIEHTLQWAR 573 conjugating system 553 UbiquitinUbe1y1; 184; 185 GIK*LVVADTR 574 conjugating UBE1 system 554 UbiquitinUBE2D3; 128; 128 IYK*TDRDKYNR 575 conjugating UBE2D4 system 555Chromatin, UBE2N 82 IYHPNVDK*LGR 576 DNA- binding, DNA repair or DNAreplication protein 556 Chromatin, UBE2N 92 ICLDILK*DKWSPALQIR 577 DNA-binding, DNA repair or DNA replication protein 557 Chromatin, UBE2N 94ICLDILKDK*WSPALQIR 578 DNA- binding, DNA repair or DNA replicationprotein 558 Ubiquitin UBE2Q1 216, 232, K*SEDDGIGKENLAILEK*IK* 579conjugating 234 system 559 Ubiquitin ubiquitin; 113; 113 VDENGK*ISR 580conjugating L0C388720 system 560 Ubiquitin ubiquitin; 113; 113YYKVDENGK*ISR 581 conjugating L0C388720 system 561 Ubiquitin ubiquitin;152; 152 CCLTYCFNK*PEDK 582 conjugating L0C388720 system 562 UbiquitinUBQLN1 53 EKEEFAVPENSSVQQFK*EEI 583 conjugating SKR system 563 Enzyme,UGP2 183 VK*IYTFNQSR 584 misc. 564 Mitochondrial uricase 118AHVYVEEVPWK*R 585 protein 565 Mitochondrial uricase 220DIVLQK*FAGPYDKGEYSPSV 586 protein QK 566 Protease USP33 227SRPGSVVPANLFQGIK*TVNP 587 TFR 567 Protease USP5 357 YVDK*LEKIFQNAPTDPTQD588 FSTQV AK 568 Protease USP5 360 YVDKLEK*IFQNAPTDPTQD 589 FSTQV AK 569Protease USP5 360 KYVDKLEK*IFQNAPTDPTQ 590 DFSTQ VAK 570 Protease USP5575 FASFPDYLVIQIKK* 591 571 Cytoskeletal utrophin 50 SGK*PPISDM#FSDLKDGR592 protein 572 Enzyme, VARS 951 HFCNK*LWNATK 593 misc. 573 Chromatin,VCP 109 LGDVISIQPCPDVK*YGKR 594 DNA- binding, DNA repair or DNAreplication protein 574 Chromatin, VCP 336 IVSQLLTLMDGLK*QR 595 DNA-binding, DNA repair or DNA replication protein 575 Chromatin, VCP 505ELQELVQYPVEHPDKFLK* 596 DNA- binding, DNA repair or DNA replicationprotein 576 Chromatin, VCP 668 KSPVAK*DVDLEFLAK 597 DNA- binding, DNArepair or DNA replication protein 577 Receptor, VDAC-1 287NVNAGGHK*LGLGLEFQA 598 channel, transporter or cell surface protein 578RNA vigilin 494 IEGDPQGVQQAK*R 599 processing 579 Unknown WDR19 1171,1185 IHVKSGDHMK*GARM#LIR 600 function VANNIS K* 580 Transcriptional YB-1168 NYQQNYQNSESGEK*NEGS 601 regulator ESAP EGQAQQR 581 TranscriptionalZNF318 1246, EVK*EDDK*APGELEEQLSE 602 regulator 1250, DGSAPEK*GEVKGNASLR 1268 % in Ubiquitinated Residue Number indicatesubiquitination sites described in scientific literature K* in PeptideSequence indicates lysine residues modified with Gly-Gly from ubiquitinor ubiquitin-like proteins, i.e., Lys-epsilon-Gly-Gly

indicates data missing or illegible when filed

This experiment resulted in the identification of 581 peptides that hadbeen modified by ubiquitin or ubiquitin-like protein and allowed for thelocalization of the specific sites of ubiquitination within thepredicate polypeptide. The experiment identified 6 of 7 knownubiquitination sites in ubiquitin itself. (See rows 39-48 of Table 4;Ikeda F, Dikic I. Atypical ubiquitin chains: new molecular signals.‘Protein Modifications: beyond the Usual Suspects’ review series. EMBORep. 2008 June; 9(6):536-42.

Additionally, novel ubiquitination sites in enzymes responsible forlinking ubiquitin to other proteins as part of the ubiquitin conjugatingsystem were discovered (see rows 550-562 in Table 4).

Neddylation sites in ubiquitin-like molecules such as NEDD8 (see row 392in Table 4) were also identified as trypsin digestion of neddylatedproteins leaves the same K(GG) remnant as trypsin digestion ofubiquitinated proteins. Thus, the invention contemplates the use of theantibodies described herein in, for example, the methods describedherein to identify neddylated proteins following digestion of suchneddylated proteins with a hydrolyzing agent such as trypsin. NEDD8 isabout 60% identical to ubiquitin and like ubiquitin can formpolyneddylation chains (Jones J, Wu K, Yang Y, Guerrero C, Nillegoda N,Pan Z Q, Huang L. A targeted proteomic analysis of the ubiquitin-likemodifier nedd8 and associated proteins. J Proteome Res. 2008 March;7(3):1274-87). Several known ubiquitination sites in histones (e.g., H2Aand H₂B; see rows 13-26 and 27-30, respectfully, in Table 4) wereidentified. Ubiquitination of these histones is thought to regulate manynuclear processes such as transcription, silencing, and DNA repair(Weake V M, Workman J L. Histone ubiquitination: triggering geneactivity. Mol. Cell. 2008 Mar. 28; 29(6):653-63).

The invention also contemplates the use of the antibodies describedherein in, for example, the methods described herein to identifyproteins modified by the Interferon-induced 17 kDa protein, also calledthe I5G15 protein because it is encoded by the I5G15 gene (see Blomstromet al., J Biol Chem 261 (19): 8811-8816, 1986). Following digestion ofsuch I5G15-modified proteins with a hydrolyzing agent such as trypsin,the antibodies of the invention will specifically bind to and recognizethe modified lysine residues in the hydrolyzed ISG15-modified proteins(see, e.g., Zhao et al., Proc. Natl. Acad. Sci. 107(5): 2253-2258,2010).

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of this invention.Although any compositions, methods, kits, and means for communicatinginformation similar or equivalent to those described herein can be usedto practice this invention, the preferred compositions, methods, kits,and means for communicating information are described herein.

All references cited above are incorporated herein by reference in theirentirety to the extent allowed by law. The discussion of thosereferences is intended merely to summarize the assertions made by theirauthors. No admission is made that any reference (or a portion of anyreference) is relevant prior art. Applicants reserve the right tochallenge the accuracy and pertinence of any cited reference.

1-17. (canceled)
 18. An isolated antibody that preferentially binds aubiquitin remnant peptide over a peptide having the same amino acidsequence as the ubiquitin remnant peptide but lacking a ubiquitinremnant, wherein the antibody comprises a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, and SEQ ID NO:
 8. 19-20. (canceled)
 21. An isolatedbinding partner that specifically binds a ubiquitin remnant peptide butdoes not specifically bind a peptide having the same amino acid sequenceas the ubiquitin remnant peptide but lacks the ubiquitin remnant.
 22. Anisolated antibody that specifically binds a ubiquitin remnant peptidebut does not specifically bind a peptide having the same amino acidsequence as the ubiquitin remnant peptide but lacks the ubiquitinremnant.
 23. The antibody of claim 22, wherein the antibody is amonoclonal antibody.
 24. The antibody of claim 22, wherein the antibodyis a polyclonal antibody.
 25. The antibody of claim 22, wherein theantibody is a human antibody.
 26. The antibody of claim 22, wherein theantibody is a chimeric antibody.
 27. The antibody of claim 22, whereinthe antibody has a dissociation constant (kD) of 10⁻⁷M or less for theubiquitin remnant peptide.
 28. The antibody of claim 22, wherein theantibody has a dissociation constant (kD) of 10⁻⁹M or less for theubiquitin remnant peptide.
 29. The antibody of claim 22, wherein theantibody has an off rate (k_(off)) of 10⁻³/sec or less.
 30. The antibodyof claim 22, wherein the antibody has an off rate (k_(off)) of 10⁻⁴/secor less.
 30. The antibody of claim 22, wherein the antibody has an offrate (k_(off)) of 10⁻⁵/sec or less.
 31. The antibody of claim 22,wherein the antibody specifically binds a ubiquitinated lysine residuein the ubiquitin remnant peptide regardless of the peptide sequencesurrounding the ubiquitinated lysine residue.
 32. The antibody of claim22, wherein the antibody is a monoclonal antibody.
 33. The antibody ofclaim 22, wherein the ubiquitin remnant is a diglycine modified epsilonamine of lysine.
 34. The antibody of claim 22, wherein the ubiquitinremnant is a portion of a ubiquitinated polypeptide that remainsattached to a digestion product of the ubiquitinated polypeptide thathas been exposed to trypsin.